Optical sources for fluorescent lifetime analysis

ABSTRACT

Compact optical sources and methods for producing short and ultrashort optical pulses are described. A semiconductor laser or LED may be driven with a bipolar waveform to generate optical pulses with FWHM durations as short as approximately 85 ps having suppressed tail emission. The pulsed optical sources may be used for fluorescent lifetime analysis of biological samples and time-of-flight imaging, among other applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/161,067 titled “Optical Sources for Fluorescent Lifetime Analysis”filed May 20, 2016, which claims priority to U.S. provisionalapplication No. 62/164,485 titled, “Pulsed Laser” filed on May 20, 2015,to U.S. provisional application No. 62/296,546, filed Feb. 17, 2016,titled “Sensor and Device for Lifetime Imaging and DetectionApplications,” and to U.S. provisional application No. 62/310,398titled, “Pulsed Laser and System” filed on Mar. 18, 2016. U.S. patentapplication Ser. No. 15/161,067 is a continuation-in-part of U.S.application Ser. No. 14/821,656, filed Aug. 7, 2015, titled “IntegratedDevice for Temporal Binning of Received Photons.” Each of the foregoingapplications is hereby incorporated by reference in its entirety. U.S.application Ser. No. 15/161,067 also claims priority to U.S. provisionalapplication No. 62/164,506 titled, “Integrated Device For TemporalBinning of Received Photons” filed on May 20, 2015 and to U.S.provisional application No. 62/164,464 titled, “Integrated Device WithExternal Light Source for Probing Detecting and Analyzing Molecules,filed on May 20, 2015.

FIELD

The present application is directed to devices and methods for producingshort and ultrashort optical pulses for time-domain applications thatinclude fluorescent lifetime and time-of-flight applications.

BACKGROUND

Ultrashort optical pulses (i.e., optical pulses less than about 100picoseconds) are useful in various areas of research and development aswell as commercial applications involving time-domain analyses. Forexample, ultrashort optical pulses may be useful for time-domainspectroscopy, optical ranging, time-domain imaging (TDI), and opticalcoherence tomography (OCT). Ultrashort-pulses may also be useful forcommercial applications including optical communication systems, medicalapplications, and testing of optoelectronic devices and materials.

Conventional mode-locked lasers have been developed to produceultrashort optical pulses, and a variety of such lasers are currentlyavailable commercially. For example, some solid-state lasers and fiberlasers have been developed to deliver pulses with durations well below200 femtoseconds. However, for some applications, these pulse durationsmay be shorter than is needed to obtain useful results, and the cost ofthese lasing systems may be prohibitively high. Additionally, theselasing systems may be stand-alone systems that have a sizeable footprint(e.g., on the order of 1 ft² or larger) and appreciable weight, and maynot be readily portable. Such lasing systems and their drivingelectronics may be difficult to incorporate into an instrument as areplaceable module, or even be incapable of being incorporated into ahand-held device. As a result, ultra-short pulsed lasers are oftenmanufactured as a separate stand-alone instrument from which an outputbeam may be coupled to another instrument for a particular application.

SUMMARY

The technology described herein relates to apparatus and methods forproducing short and ultrashort optical pulses with laser diodes (LDs) orlight-emitting diodes (LEDs). Short pulses are pulses havingfull-width-half-maximum (FWHM) temporal profiles between about 100picoseconds and about 10 nanoseconds. Ultrashort pulses are pulseshaving FWHM temporal profiles less than about 100 picoseconds.Gain-switching techniques and related circuitry are described that maybe implemented in compact, low-cost laser systems to produce pulseshaving durations less than about 2 nanoseconds in some embodiments, andless that about 100-picosecond in some cases. The inventors haverecognized and appreciated that a compact, low-cost, pulsed-laser systemmay be incorporated into instrumentation (e.g., fluorescent lifetimeimaging devices, bioanalytical instruments that utilizelifetime-resolved fluorescent detection, time-of-flight instruments,optical coherence tomography instruments) that may allow suchinstrumentation to become easily portable and produced at appreciablylower cost than is possible for such systems that use conventionalultrashort-pulsed laser systems. High portability may make suchinstruments more useful for research, development, clinical, commercial,and in-home applications.

Some embodiments relate to a pulsed optical source comprising asemiconductor diode configured to emit light, and a driving circuit thatincludes a transistor coupled to a terminal of the semiconductor diode,wherein the driving circuit is configured to receive a unipolar pulseand apply a bipolar electrical pulse to the semiconductor dioderesponsive to receiving the unipolar pulse.

Some embodiments relate to methods of producing an optical pulse. Amethod may comprise acts of receiving at least one clock signal,producing an electrical pulse from the at least one clock signal,driving a gate terminal of a transistor with the electrical pulse,wherein a current carrying terminal of the transistor is connected to asemiconductor diode that is configured to emit light, and applying abipolar current pulse to the semiconductor diode to produce an opticalpulse responsive to activation of the transistor by the electricalpulse.

Some embodiments relate to a fluorescent lifetime analysis systemcomprising a semiconductor diode configured to emit light, a drivingcircuit configured to apply a bipolar current pulse to the semiconductordiode to produce an optical pulse, an optical system arranged to deliverthe optical pulse to a sample, and a photodetector configured todiscriminate photon arrival times into at least two time bins during asingle charge-accumulation interval of the photodetector.

Some embodiments relate to a pulsed optical source comprising, asemiconductor diode configured to emit light, a first logic gateconfigured to form a first pulse at an output of the first logic gate,and a driving circuit coupled to the first logic gate, wherein thedriving circuit is configured to receive the first pulse and apply abipolar electrical pulse to the semiconductor diode to produce anoptical pulse responsive to receiving the first pulse.

Some embodiments relate to a pulsed optical source comprising asemiconductor diode configured to emit light, and a driving circuit thatincludes a transistor coupled to a terminal of the semiconductor diode,wherein the driving circuit is configured to receive a unipolar pulseand apply a bipolar electrical pulse to the semiconductor dioderesponsive to receiving the unipolar pulse, wherein the transistor isconnected in parallel with the semiconductor diode between a currentsource and a reference potential.

Some embodiments relate to a pulsed optical source comprising asemiconductor diode configured to emit light, and plural first circuitbranches connected to a first terminal of the semiconductor diode, eachcircuit branch comprising a transistor having its current-carryingterminals connected between a reference potential and the first terminalof the semiconductor diode.

Some embodiments relate to a pulsed optical source comprising aradio-frequency amplifier providing a signal and an inverted signal, alogic gate configured to receive the signal and a phase-shifted invertedsignal and output a pulse and an inverted pulse, a combiner configuredto combine the pulse and inverted pulse onto a common output, and asemiconductor diode coupled to the common output and configured toproduce an optical pulse responsive to receiving the pulse and invertedpulse.

Some embodiments relate to a pulsed optical source comprising aradio-frequency logic gate configured to receive a first signal and aninverted version of the first signal and output a pulse and an invertedversion of the pulse, and a semiconductor diode connect to theradio-frequency logic gate and arranged to receive the pulse at a firstterminal of the semiconductor diode and the inverted version of thepulse at a second terminal of the semiconductor diode and emit anoptical pulse.

The foregoing and other aspects, implementations, acts, functionalities,features and, embodiments of the present teachings can be more fullyunderstood from the following description in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

FIG. 1-1 depicts a pulsed lasing system incorporated with an analyticalinstrument, according to some embodiments.

FIG. 1-2 depicts a train of ultrashort optical pulses, according to someembodiments.

FIG. 2-1A illustrates optical pump and output pulses for gain switching,according to some embodiments.

FIG. 2-1B illustrates relaxation oscillations, according to someembodiments.

FIG. 2-1C depicts an optical output pulse showing a tail, according tosome embodiments.

FIG. 2-2A depicts a pulsed semiconductor laser diode, according to someembodiments.

FIG. 2-2B depicts a pulser circuit schematic for pulsing a laser diodeor light-emitting diode, according to one embodiment.

FIG. 2-2C illustrates improvements in current delivered to a laserdiode, according to some embodiments.

FIG. 2-3 depicts a current drive waveform for gain-switching a laserdiode, according to some embodiments.

FIG. 2-4A depicts a pulser circuit for driving a laser diode orlight-emitting diode, in some embodiments.

FIG. 2-4B depicts a pulser circuit schematic for driving a laser diodeor light-emitting diode, according to some embodiments.

FIG. 2-4C depicts a pulser circuit schematic for driving a laser diodeor light-emitting diode, according to some embodiments.

FIG. 2-4D depicts an RF driver for pulsing a laser diode orlight-emitting diode, according to some embodiments.

FIG. 2-4E illustrates a drive waveform produced by the circuit of FIG.2-4D, according to some embodiments.

FIG. 2-4F depicts an RF driver for pulsing a laser diode orlight-emitting diode, according to some embodiments.

FIG. 2-4G illustrates drive waveforms produced by the circuit of FIG.2-4F, according to some embodiments.

FIG. 2-4H depicts a pulser circuit schematic for driving a laser diodeor light-emitting diode, according to some embodiments.

FIG. 2-4I illustrates efficiency of power coupling to a laser diode,according to some embodiments.

FIG. 2-4J depicts a pulser and driver circuit for pulsing opticalemission from a laser diode or light-emitting diode, according to someembodiments.

FIG. 2-4K depicts a pulser circuit for producing a train of pulses,according to some embodiments.

FIG. 2-4L illustrates data inputs to a logic gate in a pulser circuit,according to some embodiments.

FIG. 2-4M depicts a driver circuit for driving a laser diode orlight-emitting diode with electrical pulses, according to someembodiments.

FIG. 2-5A depicts a pulser circuit for gain-switching a laser diode,according to some embodiments.

FIG. 2-5B illustrates a drive voltage from a pulser circuit, accordingto some embodiments.

FIG. 2-5C and FIG. 2-5D illustrate example measurements of ultrafastoptical pulses produced from a gain-switched laser diode, according tosome embodiments.

FIG. 2-6A depicts a slab-coupled optical waveguide semiconductor laserthat may be gain-switched or Q-switched, according to some embodiments.

FIG. 2-6B illustrates an optical mode profile in a slab-coupled opticalwaveguide laser, according to some embodiments.

FIG. 2-6C depicts an integrated, gain-switched semiconductor laser andcoupled saturable absorber, according to some embodiments.

FIG. 3-1 depicts a system for synchronizing timing of optical pulses toinstrument electronics, according to some embodiments.

FIG. 3-2 depicts a system for synchronizing timing of optical pulses toinstrument electronics, according to some embodiments.

FIG. 3-3 depicts a system for synchronizing timing of optical pulsesfrom two pulse sources to instrument electronics, according to someembodiments.

FIG. 3-4A depicts a system for synchronizing interleaved timing ofoptical pulses from two pulse sources to instrument electronics,according to some embodiments.

FIG. 3-4B depicts interleaved and synchronized pulse trains from twopulsed optical sources, according to some embodiments.

FIG. 4-1 depicts an instrument for analyzing fluorescent lifetimes of asample, according to some embodiments.

FIG. 4-2 depicts emission probabilities for fluorescent molecules havingdifferent emission lifetimes.

FIG. 4-3 depicts time-binned detection of fluorescent emission fromfluorescent molecules.

FIG. 4-4 depicts a time-binning photodetector, according to someembodiments.

FIG. 4-5A depicts multiple excitation pulses followed by fluorescentemission and corresponding binned signals, according to someembodiments.

FIG. 4-5B depicts a histogram produced from binned signals for aparticular fluorophore, according to some embodiments.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings. When describing embodiments in referenceto the drawings, directional references (“above,” “below,” “top,”“bottom,” “left,” “right,” “horizontal,” “vertical,” etc.) may be used.Such references are intended merely as an aid to the reader viewing thedrawings in a normal orientation. These directional references are notintended to describe a preferred or only orientation of an embodieddevice. A device may be embodied in other orientations.

DETAILED DESCRIPTION I. Introduction

The inventors have recognized and appreciated that conventionalultrashort-pulsed optical sources with pulse repetition rates below 1GHz are typically large, expensive, and unsuitable for many mobileapplications. For example, conventional ultrashort-pulsed lasers may notbe incorporated into compact and portable instrumentation. The inventorshave recognized and appreciated that a small, short or ultrashort-pulsedoptical source can enable new and useful devices for a wide range oftime-domain applications. Such applications include, but are not limitedto time-of-flight imaging, ranging, fluorescent and fluorescent lifetimeanalyses, biological or chemical analyses, optical coherence tomography(OCT), and medical point-of-care (POC) instrumentation. In some cases,POC instrumentation may comprise apparatus for detecting fluorescencefrom a biological sample, and analyzing the fluorescence to determine aproperty of the biological sample. A pulsed optical source may be usedto excite fluorescence in such instrumentation. The inventors haveconceived of compact, short and ultrashort-pulsed optical sources andsystems that can produce optical pulses at various wavelengths havingpulse durations below about 2 nanoseconds, and even less than 100picoseconds, according to some embodiments.

In overview, FIG. 1-1 depicts a pulsed optical source 1-110 that may beincorporated into an analytical instrument 1-100, such as a POC or OCTinstrument that excites and detects fluorescence or a time-of-flightimaging instrument. The instrument may include an optical system 1-140and an analytic system 1-160. The optical system 1-140 may include oneor more optical components (e.g., lens, mirror, optical filter,attenuator) and be configured to operate on and/or deliver opticalpulses from the optical source 1-110 to the analytic system 1-160. Theanalytic system may include one or more components (e.g., lens, mirror,optical filter, attenuator, photodetector) arranged to receive anoptical signal (e.g., fluorescence, backscattered radiation) from asample 1-170 to be analyzed and produce an electrical signalrepresentative of the received optical signal. In some embodiments, theanalytic system 1-160 may further include electronics configured toprocess the electrical signal.

According to some embodiments, the pulsed optical source 1-110 maycomprise at least one laser diode (LD) that is gain switched. In someembodiments, the pulsed optical source 1-110 may comprise at least onelight-emitting diode (LED) that is driven with short current pulses. Apulser circuit 1-112 that generates nanosecond-scale, or shorter,current pulses may be included with an analytical instrument 1-100 todrive the optical source 1-110.

When configured as a laser diode, a pulsed optical source 1-110 maycomprise a gain medium 1-105 (e.g., any suitable semiconductor junctionwhich may or may not include multiple quantum wells), and at least twocavity mirrors 1-102, 1-104 (or reflective facets of a laser diode) thatdefine ends of an optical laser cavity. In some embodiments, there maybe one or more additional optical elements in the laser cavity forpurposes of beam shaping, polarization control, wavelength selection,and/or pulse forming. Light-collecting optics may be included with alaser diode, and configured to concentrate emission from the laser diodeinto a beam. The beam from a laser diode may or may not be collimated bythe light-collecting optics. When the laser operates in gain-switchedmode, an optical pulse may build up within the laser cavity between thecavity's end mirrors 1-102, 1-104 responsive to the application of acurrent pulse through the laser's diode junction. One of the cavitymirrors 1-104 (often referred to as an output coupler) may partiallytransmit a portion of the pulse, so that an optical pulse 1-122 isemitted from the pulsed laser 1-110. When current driving pulses arerepeatedly applied to the laser diode, a train of pulses 1-122 (only oneshown) may be emitted from the laser cavity in rapid succession. Thistrain of pulses may be referred to as a laser beam that can becharacterized by a beam waist w. The laser beam may be collimated(indicated by the parallel dashed lines), partially-collimated, or maynot be collimated. The beam waist represents a transverse dimension ofthe emitted laser beam (e.g., ±1/e² values of the transverse intensityprofile for a Gaussian beam or a full-width-half-maximum (FWHM) valuefor other transverse intensity beam profiles), and may change in valuewith distance from the output coupler. The beam collimation and waistmay depend upon the laser's cavity geometry and optical properties andwhether any optical elements (e.g., collimating lenses) are includedwith the laser cavity.

When configured as a light-emitting diode, a pulsed optical source 1-110may comprise any suitable semiconductor junction that is configured toemit incoherent or partially coherent light. Light-collecting optics maybe included and arranged to concentrate emission from the LED into anoutput beam. The beam from an LED may or may not be collimated by thelight-collecting optics. When operating, an LED generates an opticalpulse of mainly spontaneously emitted photons responsive to theapplication of a current pulse across the LED junction, though somestimulated emission may be present in the output as amplifiedspontaneous emission. Typically, a spectral bandwidth emitted from anLED is on the order of 10's of nanometers, whereas a spectral bandwidthemitted from an LD may be less than two nanometers.

A characteristic wavelength emitted from an LD or LED may be selected bya choice of semiconductor materials and/or impurities added to thesemiconductor materials. Indium-phosphide-based semiconductors andalloys thereof may be used for longer wavelengths in the red andinfrared regions of the spectrum. Gallium-arsenide-phosphide-basedsemiconductors and alloys thereof may be used for shorter wavelengthsinto the yellow region of the spectrum. Aluminum-gallium-phosphide orgallium-nitride and their alloys may be used for the green and blueregions of the spectrum.

According to some embodiments, a particular semiconductor material maybe selected for a pulsed optical source 1-110 of an instrument thatexcites and detects fluorescence (e.g., a POC fluorescent lifetimeimaging instrument) to produce pulses having one or more of thefollowing characteristic wavelengths: 270 nm, 280 nm, 325 nm, 340 nm,370 nm, 380 nm, 400 nm, 405 nm, 410 nm, 450 nm, 465 nm, 470 nm, 490 nm,515 nm, 640 nm, 665 nm, 808 nm, and 980 nm. In some implementations, asemiconductor may be selected for a pulsed optical source 1-110 of aninstrument to produce pulses having a range or spectral distribution ofwavelengths falling within one of the following ranges of wavelengths:approximately 270 nm to approximately 370 nm, approximately 340 nm toapproximately 400 nm, approximately 380 nm to approximately 490 nm, andapproximately 410 nm to approximately 470 nm.

For reference, the phrase “characteristic wavelength” or “wavelength”may refer to a central or predominant wavelength within a limitedbandwidth of radiation. In some cases, it may refer to a peak wavelengthwithin a bandwidth of radiation. The phrase “characteristic energy” or“energy” may refer to an energy associated with a characteristicwavelength. The term “optical” may refer to ultraviolet, visible, nearinfrared, and short-wavelength infrared spectral bands.

In some embodiments, an optical system 1-140 may operate on a beam ofpulses 1-122 emitted from the pulsed optical source 1-110. For example,the optical system may include one or more lenses to reshape the beamand/or change the divergence of the beam. Reshaping of the beam mayinclude increasing or decreasing the value of the beam waist and/orchanging a cross-sectional shape of the beam (e.g., elliptical tocircular, circular to elliptical, etc.). Changing the divergence of thebeam may comprise increasing or decreasing the beam's divergence. Insome implementations, the optical system 1-140 may include an attenuatoror optical amplifier to change the amount of beam energy. In some cases,the optical system may include wavelength filtering elements. In someimplementations, the optical system may include pulse shaping elements,e.g., a pulse stretcher and/or pulse compressor. In some embodiments,the optical system may include one or more nonlinear optical elements,such as a saturable absorber for reducing a pulse length or a nonlinearcrystal for converting the pulse wavelength to a shorter wavelength viafrequency doubling or a longer wavelength via parametric amplification.According to some embodiments, the optical system 1-140 may include oneor more elements that alter, select, and/or control the polarization ofthe pulses from the optical source 1-110.

Although the pulsed optical source 1-110 and optical system 1-140 areshown as separate elements from the analytic system 1-160 in FIG. 1-1,the pulsed optical source and optical system may be manufactured as acompact and replaceable module that can be housed within the analyticsystem 1-160, according to some embodiments. In some embodiments, thepulser circuit 1-112 and pulsed optical source 1-110 may be integratedonto a same board (e.g., a same printed circuit board) or a samesubstrate (e.g., a same semiconductor substrate).

In various embodiments, pulses 1-122 emitted from a pulsed opticalsource may have temporal intensity profiles as depicted in FIG. 1-2. Insome embodiments, the peak intensity values of the emitted pulses may beapproximately equal, and the profiles may have a Gaussian temporalprofile, though other profiles such as a sech² profile may be possible.In some cases, the pulses may not have symmetric temporal profiles andmay have other temporal shapes. In some embodiments, gain and/or lossdynamics within the optical source 1-110 may yield pulses havingasymmetric profiles, as described below in connection with FIG. 2-1C.The duration of each pulse may be characterized by afull-width-half-maximum (FWHM) value, as indicated in FIG. 1-2.Ultrashort optical pulses may have FWHM values less than 100picoseconds. Short optical pulses may have FWHM values less thanapproximately 10 nanoseconds.

The pulses emitted from an optical source 1-110 may be spaced in time byregular intervals T, sometimes referred to as the pulse-separationinterval. In some embodiments, T may be determined by active gain and/orloss modulation rates in a laser. For example, the repetition rate atwhich a laser diode is gain-switched or current applied to the junctionof a light-emitting diode may determine the pulse-separation interval T.According to some embodiments, the pulse-separation interval T may bebetween about 1 ns and about 100 ns. In some implementations, thepulse-separation interval T may be long, for example, to repeat at aframe rate of an imaging device. In some cases, the pulse-separationinterval T may be between about 100 ns and about 50 ms.

The transverse spatial profile of pulses 1-122 may be single-modeGaussian in some embodiments, however the invention is not limited tosuch profiles. In some implementations, the transverse spatial profileof pulses 1-122 may be multi-modal, e.g., having multiple distinctintensity peaks. For a multi-mode source, the optical system 1-140 mayinclude diffusion optics that homogenize the pulses' transverseintensity profile. By allowing use of a multi-mode source, higher pulseenergies may be obtained from a laser diode. For example, the laserdiode's active region can be enlarged in a direction transverse to thelaser's optical axis to increase its optical output.

When used to excite fluorescence, pulses 1-122 from a pulsed opticalsource may be referred to as “excitation pulses.”

The term “fluorescent molecules” may be used to refer fluorescent tags,fluorescent markers that may be attached to molecular probes,fluorophores, and autofluorescent molecules. The term “fluorescence” maybe used to refer to light emitted from fluorescent tags, fluorescentmarkers that may be attached to molecular probes, fluorophores, andautofluorescent molecules.

II. Pulsed Optical Sources

The inventors have conceived of pulser circuits and techniques forproducing short and ultrashort optical pulses from laser diodes andlight-emitting diodes. The pulsing circuits and techniques have beenemployed, in some implementations, to gain-switch semiconductor lasersand produce a train of ˜85 picosecond (ps) pulses (FWHM) having peakpowers of approximately 1 W at repetition rates of up to 100 MHz (T asshort as 10 nanoseconds). In some embodiments, a unipoloar or bipolarcurrent waveform may be produced by a pulser circuit and used to drive alaser diode's gain medium in a manner to excite optical pulses andsuppress emission at the tails of the pulses. In some embodiments, aunipoloar or bipolar current waveform may be produced by a pulsercircuit and may be used to drive one or more light-emitting diodes tooutput short or ultrashort optical pulses.

For purposes of describing gain switching in laser diodes, FIGS. 2-1Athrough 2-1C are included to illustrate laser dynamics associated withgain switching. FIG. 2-1A illustrates a pump-power curve 2-110 that isrepresentative of pump power applied to a gain medium of a gain-switchedlaser, according to some embodiments. As depicted, the pump power may beapplied for a brief duration (depicted as approximately 0.6microseconds) to the gain medium in a laser cavity. For a semiconductorlaser diode, application of pump power may comprise applying a biascurrent across a p-n junction or multiple quantum wells (MQWs) of thelaser diode. The pump power pulse may be applied repetitively atregularly-spaced time intervals, for example, at a pulse-separationinterval or pulse repetition time T.

During application of the pump power pulse, optical gain in the lasercavity increases until the gain begins to exceed optical losses in thecavity. After this point, the laser may begin to lase (i.e., amplifyphotons passing through the gain medium by the process of stimulatedemission). The amplification process results in a rapid increase inlaser light and depletion of excited states in the gain medium toproduce at least one output pulse 2-130 as depicted. In someembodiments, the pump power pulse 2-110 is timed to turn off atapproximately the same time that the peak of the output pulse occurs.Turning off the pump power pulse terminates further lasing, so that theoutput pulse 2-130 quenches. In some embodiments, the output pulse 2-130may have a shorter duration than the pump pulse 2-110, as depicted inthe drawing. For example, an output pulse 2-130 produced by gainswitching may be less than ⅕ the duration of the pump pulse 2-110.

If the pump power pulse is not turned off, then the dynamics depicted inFIG. 2-1B may occur. In this case, the pump power curve (shown as pumpcurrent density) 2-140, depicted as a step function, represents currentdensity applied to a semiconductor laser. The graph shows that the gainmedium is excited by a pumping current density, which produces a carrierdensity N in the gain region of the laser diode. The pump currentdensity I of about twice a lasing threshold current density I_(th), isapplied at time t=0, and is then left on. The graph shows the increasein carrier density N for the semiconductor gain region until the opticalgain of the laser exceeds loss in the cavity. After this point, a firstpulse 2-161 builds up, depleting the carrier density and optical gain toa value less than the cavity loss, and is emitted. Subsequently, asecond pulse 2-162 builds up, depletes carrier density N, and isemitted. The build-up and depletion of carrier density repeats forseveral cycles until the laser stabilizes into continuous wave operation(e.g., after about 7 nanoseconds in this example). The cycle of pulses(pulse 2-161, pulse 2-162, and subsequent pulses) are referred to asrelaxation oscillations of the laser.

The inventors have recognized and appreciated that a challenge whengain-switching a laser to produce ultrashort-pulses is to avoiddeleterious effects of continued relaxation oscillations. For example,if a pump power pulse 2-110 is not terminated quickly enough, at least asecond optical pulse 2-162 (due to relaxation oscillation) may begin tobuild up in the laser cavity and add a tail 2-172 to a gain-switchedoutput pulse 2-170, as depicted in FIG. 2-1C. The inventors haverecognized and appreciated that such a tail can be undesirable in someapplications, such as applications aimed at distinguishing fluorescentmolecules based on fluorescent lifetimes. If the tail of an excitationpulse is not reduced sufficiently quickly, excitation radiation mayoverwhelm a detector unless wavelength filtering is employed.Alternatively or additionally, a tail on an excitation pulse maycontinue to excite a fluorescent molecule and may complicate detectionof fluorescent lifetime.

If the tail of an excitation pulse is reduced sufficiently quickly,there may be negligible excitation radiation present during fluorescentemission. In such implementations, filtering of the excitation radiationduring detection of fluorescent emission may not be needed to detect thefluorescent emission and distinguish fluorescent molecule lifetimes. Insome cases, the elimination of excitation filtering can significantlysimplify and reduce the cost of an analytic system 1-160 as well asallow a more compact configuration for the system. For example, when afilter is not needed to suppress the excitation wavelength duringfluorescent emission, the excitation source and fluorescent detector canbe located in close proximity (e.g., on a same circuit board orintegrated device, and even within microns of each other).

The inventors have also recognized and appreciated that in some cases, atail on an excitation pulse may be tolerated. For example, an analyticsystem 1-160 may have an optical configuration that easily allows forincorporation of a wavelength filter into a detection optical path. Thewavelength filter may be selected to reject excitation wavelengths, sothat a detector receives quantifiable fluorescence from a biologicalsample. As a result, excitation radiation from the pulsed optical sourcedoes not overwhelm the detected fluorescence.

In some embodiments, a fluorescent molecule's emission lifetime τ may becharacterized by a 1/e intensity value, according to some embodiments,though other metrics may be used in some embodiments (e.g., 1/e²,emission half-life, etc.). The accuracy of determining a fluorescentmolecule's lifetime is improved when an excitation pulse, used to excitethe fluorescent molecule, has a duration that is less than thefluorescent molecule's lifetime. Preferably, the excitation pulse has aFWHM duration that is less than the fluorescent molecule's emissionlifetime by at least a factor of three. An excitation pulse that has alonger duration or a tail 2-172 with appreciable energy may continue toexcite the fluorescent molecule during a time when decaying emission isbeing evaluated, and complicate the analysis of fluorescent moleculelifetime. To improve fluorescent lifetime determination in such cases,deconvolution techniques may be used to deconvolve the excitation pulseprofile from the detected fluorescence.

In some cases, it may be preferable to use ultrashort-pulses to excitefluorescent molecules in order to reduce quenching of the fluorescentmolecule or sample. It has been found that extended pumping of afluorescent molecule may bleach and/or damage the fluorescent moleculeover time, whereas higher intensities for shorter durations (even thoughfor a same total amount of energy on the molecule) may not be asdamaging to the fluorescent molecule as the prolonged exposure at lowerintensity. Reducing exposure time may avoid or reduce photo-induceddamage to fluorescent molecules, and increase the amount of time ornumber of measurements for which the fluorescent molecules may be usedin an analytic system 1-160.

In some applications, the inventors have found it desirable for theexcitation pulse to terminate quickly (e.g., within about 250 ps fromthe peak of the pulse) to a power level that is at least about 40 dBbelow the peak power level of the pulse. Some embodiments may toleratesmaller amounts of power reduction, e.g., between about 20 dB and about40 dB reduction within about 250 ps. Some embodiments may requiresimilar or higher amounts of power reduction within about 250 ps, e.g.,between about 40 dB and about 80 dB in some embodiments, or betweenabout 80 dB and about 120 dB in some embodiments. In some embodiments,these levels of power reduction may be required within about 100 ps fromthe peak of the pumping pulse.

According to some embodiments, the pulse-separation interval T (see FIG.1-2) may also be an important aspect of a pulsed laser system. Forexample, when using a pulsed laser to evaluate and/or distinguishemission lifetimes of fluorescent molecules, the time between excitationpulses is preferably longer than any emission lifetime of the examinedfluorescent species in order to allow for sufficiently accuratedetermination of an emission lifetime. For example, a subsequent pulseshould not arrive before an excited fluorescent molecule or ensemble offluorescent molecules excited from a previous pulse has (or have) had areasonable amount of time to fluoresce. In some embodiments, theinterval T needs to be long enough to determine a time between anexcitation pulse that excites a fluorescent molecule and a subsequentphoton emitted by the fluorescent molecule after termination ofexcitation pulse and before the next excitation pulse.

Although the interval between excitation pulses T should be long enoughto determine decay properties of the fluorescent species, it is alsodesirable that the pulse-separation interval T is short enough to allowmany measurements to be made in a short period of time. By way ofexample and not limitation, emission lifetimes (1/e values) offluorescent molecules used in some applications may be in the range ofabout 100 picoseconds to about 10 nanoseconds. Therefore, depending onthe fluorescent molecules used, a pulse-separation interval as short asabout 200 ps may be used, whereas for longer lifetime fluorescentmolecules a pulse-separation interval T greater than about 20nanoseconds may be used. Accordingly, excitation pulses used to excitefluorescence for fluorescent lifetime analysis may have FWHM durationsbetween about 25 picoseconds and about 2 nanoseconds, according to someembodiments.

In some applications, such as fluorescent lifetime imaging, where anintegrated time-domain imaging array is used to detect fluorescence andprovide data for lifetime analysis and a visual display, thepulse-separation interval T may not need to be shorter than a frame rateof the imaging system. For example, if there is adequate fluorescentsignal following a single excitation pulse, signal accumulation overmultiple excitation pulses for an imaging frame may not be needed. Insome embodiments, a pulse repetition rate R_(p) of the pulsed opticalsource 1-110 may be synchronized to a frame rate R_(f) of the imagingsystem, so that a pulse repetition rate may be as slow as about 30 Hz.In other embodiments, the pulse repetition rate may be appreciablyhigher than the frame rate, and fluorescent decay signals for each pixelin an image may be integrated values following multiple excitationpulses.

An example of a pulsed optical source 2-200 is depicted in FIG. 2-2A.According to some embodiments, a pulsed optical source 2-200 maycomprise a commercial or custom semiconductor laser diode 2-201 (or oneor more LEDs) formed on a substrate 2-208. A laser diode or LED may bepackaged in a housing 2-212 that includes an electrical connector 2-224.There may be one or more optical elements 2-205 (e.g., one or morelenses) included with the package to reshape and/or change thedivergence of an output beam from the laser or LED. The laser diode2-201 (or one or more LEDs) may be driven by a pulser circuit 2-210which may provide a sequence of current pulses over a connecting cable2-226 and at least one wire 2-220 to the diode 2-201. The drive currentfrom the pulser circuit 2-210 may produce a train of optical pulses2-222 emitted from the laser diode or LED.

One advantage of using LEDs is their lower cost compared to laserdiodes. Additionally, LEDs provide a broader, typically incoherent,spectral output that can be better suited for imaging applications(e.g., an LED may produce less optical interference artifacts). For alaser diode, the coherent radiation can introduce speckle unlessmeasures are taken to avoid speckle in the collected images. Also, LEDscan extend excitation wavelengths into the ultraviolet (e.g., down toabout 240 nm), and can be used for exciting autofluorescence inbiological samples.

According to some embodiments, a laser diode 2-201 may comprise asemiconductor junction comprising a first layer 2-202 having a firstconductivity type (e.g., p-type) and a second layer 2-206 having anopposite conductivity type. There may be one or more intermediate layers2-204 formed between the first and second layers. For example, theintermediate layers may comprise multiple-quantum-well (MQW) layers inwhich carriers injected from the first and second layers recombine toproduce photons. In some embodiments, the intermediate layers mayinclude electron and/or hole blocking layers. The laser diode maycomprise inorganic materials and/or organic semiconductor materials insome implementations. The materials may be selected to obtain a desiredemission wavelength. For example and for inorganic semiconductors,III-nitride compositions may be used for lasers emitting at wavelengthsless than about 500 nm, and III-arsenide or III-phosphide compositionsmay be used for lasers emitting at wavelengths greater than about 500nm. Any suitable type of laser diode 2-201 may be used including, butnot limited to, a vertical cavity surface emitting laser (VCSEL), anedge-emitting laser diode, or a slab-coupled optical waveguide laser(SCOWL).

According to some embodiments, one or more LEDs may be used instead of alaser diode. An LED may have a lower intensity than a LD, so multipleLEDs may be used. Because an LED does not undergo relaxationoscillations or dynamics associated with lasing action, its outputpulses may be of longer duration and have a wider spectral bandwidththan would occur for a laser. For example, the output pulses may bebetween about 50 ps and about 2 ns, and the spectral bandwidth may beabout 20 nm or larger. In some implementations, output pulses from anLED may be between about 100 ps and about 500 ps. Longer excitationpulses may be acceptable for fluorescent molecules having longer decaytimes. Additionally, an LED may produce an unpolarized or partiallypolarized output beam. The embodiments of pulser circuits describedbelow may be used to drive one or more LEDs in some implementations ofpulsed optical sources.

The inventors have recognized that some conventional laser diode systemscomprise current driver circuitry that can be modeled as depicted inFIG. 2-2B. For example, the current driver 2-210 may comprise a pulsedvoltage source 2-230 configured to deliver current pulses to a laserdiode. Connection to the laser diode is typically made through a cable2-226, adaptor or connector 2-224, and a single wire 2-220 that isbonded to a contact pad on the laser diode 2-210. The connection betweenthe adaptor 2-224 and laser diode may include a series inductance L1 andseries resistance R1. The connection may also include small junctioncapacitances (not shown) associated with contacts and/or the diodejunction.

The inventors have recognized and appreciated that increasing the numberof wire bonds (e.g., between the connector 2-224 and laser diode 2-201)may reduce the inductance and/or resistance of the connection to a laserdiode 2-201. Such a reduction in inductance and/or resistance may enablehigher speed current modulation of the laser diode and shorter outputpulses. According to some embodiments, a single wire bond 2-220 may bereplaced with multiple parallel wire bonds to improve the speed of alaser diode. For example, the number of wire bonds may be increased tothree or more. In some implementations, there may be up to 50 wire bondsto a laser diode.

The inventors have investigated the effects of increasing the number ofwire bonds 2-220 on a commercial laser diode. An example commerciallaser considered was an Oclaro laser diode, model HL63133DG, nowavailable from Ushio, of Cypress, Calif. Results from numericalsimulations of increasing a number of wire bonds are illustrated in FIG.2-2C. The simulation increased the number of wire bonds from a singlebond for the commercial device (curve 2-250) to three wire bonds (curve2-252) and to 36 wire bonds (curve 2-254). The average drive currentdelivered to the laser diode for a fixed 18V pulse was determined over arange of frequencies for the three different cases. The results indicatethat a higher number of wire bonds allows more current to be deliveredto the laser diode at higher frequencies. For example, at 1 GHz, the useof just three wire bonds (curve 2-252) allows more than four times asmuch current to be delivered to the laser diode than for a single wirebond. Since short and ultrashort pulses require higher bandwidth (higherfrequency components to form the short pulse), adding multiple wirebonds allows the higher frequency components to drive the laser diode ina shorter pulse than a single wire bond. In some implementations, themultiple wire bonds may extend between a single contact pad or multiplecontact pads on a laser diode and an adaptor or connector 2-224 on alaser diode package. The connector may be configured for connection toan external, standardized cable (e.g., to a 50-ohm BNC or SMA cable).

In some embodiments, the number of wire bonds and the wire bondconfiguration may be selected to match an impedance of the adaptorand/or cable connected to the laser diode. For example, the impedance ofthe wire bonds may be matched to the impedance of a connector 2-224 toreduce power reflections from the laser diode to the current driver,according to some embodiments. In other embodiments, the impedance ofthe wire bonds may be selectively mismatched to generate a negativepulse between positive current-driving pulses. Selecting a packagingmethod for a laser diode (e.g., selecting a number of wire bonds to alaser diode from an adaptor) may improve the current modulation suppliedto the laser diode at higher frequencies. This can make the laser diodemore responsive to high-speed gain-switching signals, and may enableshorter optical pulses, faster reduction of optical power after thepulse peak, and/or increased pulse repetition rates.

Referring now to FIG. 2-3, the inventors have further recognized andappreciated that applying a bipolar pulse waveform 2-300 to a laserdiode may suppress an undesired emission tail 2-172 (see FIG. 2-1C) onproduced optical pulses. A bipolar pulse may also be used to shorten anoptical pulse from an LED. A bipolar pulse may comprise a first pulse2-310 of a first polarity followed by a second pulse 2-312 of anopposite polarity. The magnitude of the second pulse 2-312 may bedifferent from the magnitude of the first pulse. In some embodiments,the second pulse may have a magnitude that is approximately equal to orless than the first pulse 2-310. In other embodiments, the second pulse2-312 may have a magnitude that is greater than the first pulse 2-310.

In some embodiments, the magnitude of the second pulse may be betweenabout 10% of the magnitude of the first pulse and about 90% of themagnitude of the first pulse. In some implementations, the magnitude ofthe second pulse may be between about 25% of the magnitude of the firstpulse and about 90% of the magnitude of the first pulse. In some cases,the magnitude of the second pulse may be between about 50% of themagnitude of the first pulse and about 90% of the magnitude of the firstpulse. In some embodiments, an amount of energy in the second pulse maybe between about 25% of an amount of energy in the first pulse and about90% of the energy in the first pulse. In some implementations, an amountof energy in the second pulse may be between about 50% of an amount ofenergy in the first pulse and about 90% of the energy in the firstpulse.

The first drive pulse may forward bias a laser diode junction andthereby generate carriers in the diodes active region that may recombineto produce an optical pulse. The second drive pulse 2-312, opposite inpolarity, may reverse bias the diode junction and accelerate removal ofcarriers from the active region to terminate photon generation. When thesecond electrical pulse 2-312 is timed to occur at approximately thesame time as, or just before (e.g., within about 200 ps), the secondrelaxation oscillation pulse (see pulse 2-162 of FIG. 2-1B), the carrierconcentration that would otherwise produce the second optical pulse isdiminished so that the emission tail 2-172 is suppressed.

Various circuit configurations may be used to produce bipolar pulsewaveforms. FIG. 2-4A depicts just one example of a circuit that may beused to drive a laser diode or one or more LEDs with a bipolar pulsewaveform. In some embodiments, a transmission line 2-410 (e.g., a stripline or co-axial conductor assembly) may be configured in a pulsercircuit 2-400 to deliver bipolar pulses to a semiconductor laser diode2-420 or at least one LED. The transmission line 2-410 may be formed ina U-shaped configuration and biased on a first conductor by a DC voltagesource V_(DD) through a charging resistor R_(ch). The transmission linemay have an impedance that approximately matches the impedance of alaser diode, according to some embodiments. In some embodiments, thetransmission line's impedance may be approximately 50 ohms. In someimplementations, the transmission line's impedance may be betweenapproximately 20 ohms and approximately 100 ohms. In someimplementations, the transmission line's impedance may be betweenapproximately 1 ohm and approximately 20 ohms.

The pulser 2-400 may further include a terminating resistor Z_(term)connected between the second conductor of the transmission line at oneend of the transmission line and a reference potential (e.g., ground inthe depicted example). The other end of the second conductor of thetransmission line may be connected to the laser diode 2-420. The ends ofthe transmission line's first conductor may connect to a switch M1(e.g., a field effect transistor or bipolar junction transistor) thatcan be activated to periodically shunt the ends of the first conductorto a reference potential (e.g., ground).

In some instances, the terminating impedance Z_(term) may beapproximately equal to the impedance of the transmission line 2-410 inorder to reduce reflections back into the line. Alternatively, theterminating impedance Z_(term) may be less than the impedance of theline in order to reflect a negative pulse into the line (after shuntingby switch M1) and to the laser diode 2-420. In some implementations, theterminating impedance Z_(term) may include a capacitive and/or inductivecomponent selected to control the shape of the reflected negative pulse.A transmission line pulser, as depicted in FIG. 2-4A, may be used toproduce electrical bipolar pulses having a repetition rate within arange between about 30 Hz to about 200 MHz. According to someembodiments, a transmission line 2-410 for a transmission line pulsermay be formed on a printed circuit board (PCB), as depicted in FIG.2-5A.

FIG. 2-4B depicts an embodiment of a driver circuit 2-401 connected toan optical semiconductor diode 2-423 (e.g., a laser diode or one or moreLEDs) that may be formed using discrete components, and that may beintegrated onto a substrate (such as a chip or PCB). In someembodiments, the circuit may be integrated onto a same substrate as alaser diode or LED 2-423. The laser driver circuit 2-401 may comprise acontrol input 2-405 connected to the gate or base of a transistor M1.The transistor may be a CMOS FET, a bipolar junction transistor, or ahigh-electron mobility transistor (such as a GaN pHEMT), though otherhigh-speed, high current handling transistors may be used. Thetransistor may be connected between a current source 2-430 and areference potential (e.g., a ground potential, though other referencepotential values may be used). The transistor M1 may be connected inparallel between the current source 2-430 and reference potential withthe laser diode 2-423 (or one or more LEDs) and a resistor R₁ that isconnected in series with the laser diode. According to some embodiments,the driver circuit 2-401 may further include a capacitor C₁ connected inparallel with the resistor R₁ between the laser diode and referencepotential. Though a transistor M1 is described, any suitablecontrollable switch having a high conductive and low conductive statemay be used.

In operation, the driver circuit 2-401 may provide a current thatbypasses the laser diode 2-423 when the transistor M1 is on, or in aconducting state. Therefore, there is no optical output from the laserdiode. When the transistor M1 switches off, current may flow through thelaser diode due to the increased resistive path at the transistor. Thecurrent turns the laser diode on, until the transistor is switched onagain. Light pulses may be generated by modulating the control gate ofthe transistor between on and off states to provide current pulses tothe laser diode. This approach can reduce the amount of voltage on thesupply and the voltage on the transistor needed to drive the lasercompared to some pulsing techniques, which is an important aspect forimplementation of such high-speed circuits.

Due to the presence of the resistor R₁ and parallel capacitor C₁, chargewill build up on the capacitor when the diode is forward conducting.This can occur when the transistor M1 is in an “off” state, e.g., a low-or non-conducting state. When the transistor is turned on, the voltagestored across the capacitor will reverse bias the laser diode. Thereverse bias effectively produces a negative pulse across the laserdiode, which may reduce or eliminate the emission tail 2-172 that wouldotherwise occur without the negative pulse. The value of the resistor R₁may be selected such that substantially all of the charge on thecapacitor will discharge before the switch is subsequently opened and/ora subsequent light pulse is generated by the laser diode. For example,the time constant t₁=R₁C₁ may be engineered to be less than aboutone-half or one-third of the pulse repetition interval T. In someimplementations, the time constant t₁=R₁C₁ may be between approximately0.2 ns and approximately 10 ns.

In some implementations, the transistor M1 may be configured to switchto a conducting state after a first peak of an output light pulse fromthe laser diode. For example, and referring to FIG. 2-1B, an opticaldetection and logic circuit may sense the decaying intensity of thefirst pulse 2-161 and trigger the transistor M1 to switch to aconducting state. In some embodiments, the transistor M1 may betriggered to switch to a conducting state based on a stable clock signal(e.g., triggered with reference to a synchronizing clock edge). In someimplementations, the transistor M1 may be triggered to switch to aconducting state according to a predetermined delay time measured fromthe time at which the transistor M1 switches to a non-conducting state.Switching the transistor M1 to a conducting state at a selected time mayreduce the laser power shortly after the peak light pulse, shorten thelaser pulse, and/or reduce tail emission of the pulse.

Although the drive circuit shown in FIG. 2-4B shows the current source2-430 located on the anode side of the laser, in some embodiments acurrent source may be located alternatively, or additionally, on thecathode side of the laser (e.g., connected between the transistor M1,resistor R₁, and a reference potential such as ground).

Other embodiments of drive circuitry for producing ultrashort-pulses arepossible. For example, a current pulse drive circuit 2-402 for a laserdiode or LED may comprise a plurality of current drive branchesconnected to a node of a laser diode, as depicted in FIG. 2-4C. Thedriver circuit 2-402 may be formed using discrete or integratedcomponents and integrated onto a substrate (e.g., an ASIC chip or PCB).In some embodiments, the driver circuit may be integrated onto a samesubstrate as one or more optical semiconductor diodes 2-425 (e.g., alaser diode or one or more light-emitting diodes). Although the drawingdepicts the driver circuit as connected to the anode of the laser diode2-425, in some embodiments similar drive circuitry may alternatively, oradditionally, be connected to the cathode of the laser diode. Drivecircuitry connected to the cathode side of the laser diode may employtransistors of an opposite type and voltage sources of opposite polaritythan those used on the anode side of the laser diode.

According to some implementations, there may be N circuit branches(e.g., circuit branches 2-432, 2-434, 2-436) configured to apply Nforward-bias current pulses to a laser diode 2-425 or LED and M circuitbranches (e.g., circuit branch 2-438) configured to apply M reverse-biascurrent pulses to the laser diode. In FIG. 2-4C, N=3 and M=1, thoughother values may be used. Each forward-bias current branch may comprisea voltage source V_(i) configured to deliver a forward-bias current tothe laser diode. Each reverse-bias current branch may comprise a voltagesource V_(j) configured to deliver a reverse-bias current to the laserdiode. Each circuit branch may further include a resistor R_(i)connected in series with a switch or transistor Mi. Each circuit branchmay include a capacitor C_(i) connected on one side to a node betweenthe transistor Mi and resistor R_(i) and connected on the other side toa fixed reference potential. In some embodiments, the capacitance C_(i)may be junction capacitance associated with the transistor Mi (e.g,source-to-body capacitance), and a separate discrete capacitor may notbe provided. In some implementations, at least one additional resistormay be included in series with the diode 2-425 to limit the amount oftotal current delivered from the circuit branches.

In operation, timed and pulsed control signals may be applied to thecontrol inputs S_(i) of the switches or transistors Mi, so as togenerate a sequence of current pulses from each of the circuit branchesthat are summed and applied across the laser diode junction. The valuesof components in each branch (V_(i), V_(j), R_(i), C_(i)) and the timingand pulse duration of control pulses applied to the control inputs S_(i)can be independently selected to produce a desired bipolar current pulsewaveform that is applied to the laser diode 2-425. As just one example,the values of V₁, V₂, and V₃ may be selected to have different values.The values of R₁, R₂, and R₃ may be the same, and the values of C₁, C₂,and C₃ may be the same. In this example, the staggering of pulsedsignals to the control inputs S_(i) may produce a staggered sequence ofoverlapping current pulses from the forward-bias circuit branches thathave similar pulse durations but different pulse amplitudes. A timedpulse from the reverse-bias circuit branch may produce a current pulseof opposite polarity that can quench or rapidly turn off theforward-biasing pulse, and may further produce a reverse-biasing pulsethat can suppress tail emission from the laser diode. Thereverse-biasing pulse may be timed carefully, so that it at leastpartially overlaps temporally with one or more of the forward-biasingpulses. Accordingly, the circuit depicted in FIG. 2-4C may be used tosynthesize bipolar current pulses as depicted in FIG. 2-3.

FIG. 2-4D depicts another embodiment of a pulse driver 2-403, which maybe manufactured using radio-frequency (RF) components. The RF componentsmay be designed to handle signals at frequencies between about 50 MHzand about 1 GHz, according to some embodiments. In some implementations,a pulse driver 2-403 may comprise an input DC block 2-435, which ACcouples an input waveform (e.g., a square wave or sinusoidal wave) tothe driver. The DC block may be followed by an amplifier 2-440, whichproduces non-inverted and inverted output waveforms that proceed alongseparate circuit paths 2-440 a, 2-440 b, respectively. The first circuitpath 2-440 a may include one or more adaptors 2-442. A variable phaseshifter 2-445 may be included in the second circuit path 2-440 b toselectively phase shift the signal in the second path with respect tothe signal in the first path.

The first and second circuit paths may connect to non-inverting inputsof an RF logic gate 2-450 (e.g., an AND gate or other logic gate).Inverting inputs of the logic gate 2-450 may be terminated with suitableimpedance-matched terminators 2-446 to avoid spurious power reflectionsat the gate. The non-inverting and inverting outputs of the logic gate2-450 may connect to a combiner 2-460 along two circuit paths 2-450 a,2-450 b. The inverted circuit path 2-450 b may include a delay element2-454 and attenuator 2-456, either or both of which may be adjustable.The delay element may be used to delay the inverted signal with respectto the non-inverted signal, and the attenuator may be used to adjust theamplitude of the inverted signal.

The resulting inverted signal and non-inverted signal from the logicgate may then be summed at the combiner 2-460. The output from thecombiner 2-460 may be connected to an RF amplifier 2-470 that providesoutput bipolar pulses to drive a laser diode or one or more LEDs. Theoutput bipolar pulses may have a waveform as depicted in FIG. 2-4E.

In operation, an input square wave or sinusoidal wave may be AC coupledinto the driver and split into the two circuit paths 2-440 a, 2-440 b asnon-inverted and inverted versions. The first amplifier 2-440 may be alimiting amplifier that squares up a sinusoidal waveform, according tosome embodiments. In the second circuit path 2-440 b the invertedwaveform may be phase shifted with an adjustable phase shifter 2-445 totemporally delay the inverted waveform with respect to the non-invertedwaveform. The resulting waveforms from the first amplifier 2-440 maythen be processed by the RF logic gate 2-450 (e.g., an AND gate) toproduce short RF pulses at the non-inverting and inverting outputs ofthe logic gate. The duration of the short RF pulses may be adjustedusing the phase shifter 2-445, according to some embodiments. Forexample, the phase shifter may adjust a time period during which boththe non-inverted waveform and inverted waveform at the input to a logicAND gate 2-450 are simultaneously in an “on” state, which will determinethe length of the output pulses.

Referring to FIG. 2-4E, the short inverted pulses 2-417 from the logicgate 2-450 may be delayed an amount δ by the delay element 2-454 withrespect to the non-inverted pulses 2-415 and attenuated by attenuator2-456 to a desired amplitude before being combined with the non-invertedpulse. In some embodiments, the negative-pulse magnitude |V_(p−)| may beless than the positive-pulse amplitude V_(p+). The pulse-separationinterval T may be determined by the frequency of the sinusoidal orsquare wave input into the pulse driver 2-403. The output pulse waveformmay, or may not, include a DC offset. Although the output waveform isdepicted as having a square-shaped waveform, capacitances andinductances in the RF components and/or cabling may produce outputpulses having more rounded waveforms, more like the waveform depicted inFIG. 2-3.

As mentioned earlier in connection with FIG. 2-4C and FIG. 2-4B, theapplication of current or voltage to a laser diode or LED can be to boththe anode and cathode of a diode in some embodiments. A radio-frequencypulse driver circuit 2-404 that can apply a split or differentialvoltage or current pulse to both the cathode and anode of a diode isdepicted in FIG. 2-4F. The front end of the circuit may be similar tothe front end of the pulse driver circuit 2-403 depicted in FIG. 2-4D,according to some embodiments. However, in the pulse driver circuit2-404 the non-inverted and inverted outputs from the logic gate 2-450may not be combined and instead applied as a differential drive to theanode and cathode of the laser diode. For simplification, the circuitryassociated with producing a subsequent negative or reverse biasing pulseis not shown in FIG. 2-4F.

An example of a split or differential drive produced by the differentialpulse driver circuit 2-404 is depicted in FIG. 2-4G. A first output fromthe logic gate 2-450 may produce a positive pulse 2-416 of amplitude+V_(p), and a second inverted output from the logic gate 2-450 mayproduce a negative pulse 2-418 of opposite amplitude −V_(p). The pulsetrains may, or may not, have a small DC offset in some embodiments. Thepresence of the positive pulse 2-416 and negative pulse 2-418 produce aforward biasing pulse across the laser diode having an effectiveamplitude 2V_(p). By splitting the bias across the laser diode andapplying a partial bias to the anode and to the cathode, the amplitudeof voltage pulses handled by the pulse driver 2-404 may be effectivelyreduced by a factor of 2. Accordingly, the pulse driver 2-404 mayoperate at a higher frequency and produce shorter pulses than it mightotherwise be able to achieve for higher amplitude pulses. Alternatively,a pulse driver circuit 2-404 may effectively double the amplitude of thedriving pulse applied across a laser diode compared to a driving circuitthat only provides a biasing pulse +V_(p) to the anode of the laserdiode. In such embodiments, the power output from the laser diode may beincreased.

Another way in which power applied to the laser diode and/or drivingspeed may be increased is depicted in FIG. 2-4H. According to someembodiments, a plurality of pulse-driver outputs 2-470 may be connectedto an anode of a laser diode 2-425 or LED. In this example, four pulsedrivers are connected to the anode of the laser diode. In someembodiments, in which differential pulse driver circuitry is used, theremay be multiple drivers connected to the cathode of the laser diode aswell. Each driver and its associated cabling may have an impedance Z₀,and a laser diode 2-425 may have been impedance Z_(L). Because of theirparallel connection, the output impedances of the drivers are divided bythe number of drivers connected to the laser diode. The power deliveredinto the diode may be increased when the combined impedances of thepulse drivers is approximately matched to the impedance of the laserdiode 2-425, or vice versa.

The graph in FIG. 2-4I illustrates the increase in efficiency of powercoupled into the laser diode 2-425 for four driving sources as afunction of the impedance of the laser diode and the laser diodecircuit. In the example, the four pulse drivers each have a lineimpedance of about 50 ohms and are configured to deliver an output pulseof 5 V amplitude with a maximum current of approximately 100 mA. Theplot shows that the power coupled into the laser diode reaches a maximumwhen the laser diode's impedance is at approximately 10 ohms. This valueis approximately equal to the parallel output impedance of the fourpulse driver outputs 2-470. Accordingly, the impedance of the laserdiode 2-425 and its associated circuitry may be designed toapproximately match the combined impedance of one or more pulse driversused to drive the laser diode, according to some embodiments.

Other circuit driver configurations may be used to pulse laser diodes orlight-emitting diodes. According to some embodiments, a currentinjection into a light-emitting diode may be pulsed to producesub-nanosecond pulses using a pulser circuit described in “A simplesub-nanosecond ultraviolet light pulse generator with high repetitionrate and peak power,” authored by P. H. Binh et al., Rev. Sci. Instr.Vol. 84, 083102 (2013), or in “An ultraviolet nanosecond light pulsegenerator using a light emitting diode for test of photodetectors”authored by T. Araki et al., Rev. Sci. Instr. Vol. 68, 1365 (1997).

Another example of a pulser circuit is depicted in FIG. 2-4J. Accordingto some embodiments, a pulser circuit may comprise a pulse generator2-480, which may receive one or more clock signals from a system clock,for example, and output a train of electrical pulses to a driver circuit2-490 that injects current pulses into a laser diode or light-emittingdiode responsive to the received electrical pulses from the pulsegenerator. Accordingly, the output optical pulses may be synchronized tothe system clock. The system clock may also be used to operate detectionelectronics (e.g., an imaging array).

According to some embodiments, the pulse generator 2-480 may be formedfrom a combination of passive and digital electronic components, and maybe formed on a first circuit board. In some cases, a pulse generator mayinclude analog circuit components. In other embodiments, a portion ofthe pulse generator may be formed on a same board as the driver circuit2-490, and a portion of the pulse generator may be formed on a separateboard remote from the driver circuit. The driver circuit 2-490 may beformed from passive, analog, and digital electronic components, and maybe formed on a same or different circuit board as the pulse generator orportion of the pulse generator. An optical source (laser diode orlight-emitting diode) may be included on a circuit board with the drivercircuit, or may be located in a system and connected to the drivercircuit 2-490 by high-speed cabling (e.g., SMA cables). In someimplementations, the pulse generator 2-480 and driver circuit 2-490 mayinclude emitter-coupled logic elements. According to some embodiments,the pulse generator 2-480, driver circuit 2-490, and opticalsemiconductor diode 2-423 may be integrated onto a same printed circuitboard, laminate, or integrated circuit.

An example of a pulse generator 2-480 is depicted in FIG. 2-4K. In someimplementations, a pulse generator may include a first stage thatproduces two differential clock outputs, one delayed with respect to theother. The first stage may receive a clock input and include a fan-out2-481 and delay 2-483. The fan-out may comprise logic drivers and logicinverters arranged to produce two copies of the clock signal and twoinverted copies of the clock signal. According to some embodiments, theclock may have a symmetric duty cycle, though asymmetric duty cycles maybe used in other embodiments. One copy and one inverted copy may form adifferential clock output (CK1, CK1 ) and may be delayed by a delayelement 2-483 with respect to a second copy and second inverted copy(CK2, CK2 ). The delay element may comprise any suitable variable orfixed delay element. Examples of delay elements include RF delay linesand logic gate delays. In some implementations, the first pair of clocksignals (CK1, CK1 ) is delayed at least a fraction of a clock cycle withrespect to the second pair of clock signals (CK2, CK2 ). A delay mayinclude one or more full cycles in addition to a fractional cycle.Within each pair of clock signals, the inverted signal may besynchronized to its counterpart so that rising and falling edges of theclocks occur at essentially the same time.

The inventors have found that ultrashort pulsing of a laser diode or LEDcan be controlled more reliably by adjusting a length of acurrent-driving pulse from the pulse generator 2-480 and maintaining afixed amplitude rather than adjusting an amplitude of an ultrashortcurrent-driving pulse. Adjusting the length of the current-driving pulseadjusts an amount of energy delivered to the laser diode per pulse. Insome embodiments, high-speed circuits allow for high-resolution controlof signal phase (e.g., by adjusting a delay or phase with an analog ordigital delay element 2-483), which can be used to obtainhigh-resolution control of pulse length, according to someimplementations.

In some cases, the first stage of the pulse generator 2-480 may comprisea dual-output clock instead of the fan-out 2-481 and delay 2-483. Adual-output clock may generate two differential clock signals, andprovide adjustable phase delay between the two differential clocksignals. In some implementations, the adjustable phase delay may have acorresponding time resolution as little as 3 ps.

Regardless of how the delayed clock signals CK1, CK2 and their inversesare produced, the signals may be transmitted over high-speedtransmission lines to a high-speed logic gate 2-485. For signaltransmission over cables between boards, the clock pulses maydeteriorate due to cabling. For example, limited bandwidth oftransmission lines may distort the clock pulses differently and resultin unequal timing. In some implementations, a same type of cabling ortransmission line may be used for all the clock signals, so thattransmission distortions affect the four clock signals equally. Forexample, when signal distortions and timing offsets are essentially thesame for the four clock signals, a resulting driving pulse produced bythe receiving logic gate 2-485 will be essentially the same as it wouldbe if there were no signal distortions from transmission of the clocksignals. Accordingly, transmission of clock signals over distances ofseveral feet may be tolerated without affecting the driving-pulseduration. This can be useful for producing ultrashort driving pulsesthat are synchronized to a system clock and have finely adjustable pulseduration (e.g., adjustable in increments of about 3 ps). If the clocksignals are produced locally (e.g., on a same board as the drivercircuit 2-490), signal distortions associated with transmission of theclock signals may not be significant and the transmission lines maydiffer to some extent.

According to some embodiments, the clock signals may be AC coupled withcapacitors C₁ and provided to data inputs of a high-speed logic gate2-485. Capacitors C₁ may have a capacitance between about 10 nF andabout 1 μF. According to some embodiments, the logic gate may comprisean emitter-coupled logic (ECL), two-input, differential AND/NAND gate.An example of logic gate 2-485 includes model MC100EP05 available fromON Semiconductor of East Greenwich, R.I. The AC-coupled signals at thedata inputs to the logic gate may appear similar to the signals depictedin FIG. 2-4L, where the horizontal dashed line indicates a zero voltagelevel. The depictions in FIG. 2-4L do not include distortions introducedby transmission lines. The distortions may round and alter the shapes ofthe signal profiles, but may not affect the relative phases of the clocksignals when a same type and length of cabling is used for each clocksignal. Delay element 2-483 may provide a delay Δt indicated by thevertical dashed lines, which may be adjustable in increments as small as3 ps. In some implementations, a delay element 2-483 may provide anadjustable delay in increments having a value between 1 ps and 10 ps.Logic gate 2-485 may process the received clock signals and produce anoutput signal at an output port Q corresponding to the delay introducedby delay element 2-483. With a small delay, the output comprises asequence of short or ultrashort pulses. With a high-speed logic gate2-485, the pulse durations may be between about 50 ps and about 2 ns(FWHM) in some embodiments, between about 50 ps and about 0.5 ns in someembodiments, between about 50 ps and about 200 ps in some embodiments,and yet between about 50 ps and about 100 ps in some embodiments. Thedriving pulses from port Q may have a substantially square profile dueto high-speed slew rates of the ECL logic gate 2-485. A biasing circuit2-487 may be connected to the output port Q, and a voltage V₁ appliedfor positive emitter-coupled logic. Output pulses provided from anoutput terminal P_(out) of the pulse generator 2-480 may include a DCoffset, according to some embodiments.

In some implementations, two or more high-speed logic gates 2-485 may beconnected in parallel between capacitors C₁ and the bias circuit 2-487.The logic gates may be the same, and operate in parallel to providegreater current driving capability at an output of the pulse generator.The inventors have recognized and appreciated that the logic gate 2-485,or gates, need to provide high speed switching (i.e., fast rise and falltimes to produce ultrashort driving pulses), and need to provide enoughoutput current to drive a high current transistor M1 in the drivercircuit 2-490. In some implementations, connecting logic gates 2-485 inparallel provides improved performance of the pulser circuit, and allowsproduction of sub-100-ps optical pulses.

FIG. 2-4M depicts an embodiment of a driver circuit 2-490, which may beconnected to a laser diode or LED 2-423. A driver circuit may include anAC-coupled input, having a capacitor C₂ in series with a resistor R₃,connected to a gate of a high-speed transistor M1. Capacitance of C₂ maybe between approximately 0.1 μF and approximately 10 μF, according tosome embodiments, and R₃ may have a value between approximately 10 ohmsand approximately 100 ohms. Transistor M1 may comprise ahigh-electron-mobility field-effect transistor (HEMT FET) capable ofswitching high currents (e.g., at least one ampere and, in some cases,up to four amps or more), according to some embodiments. Transistor M1may be a high-speed transistor capable of switching such large currentsat multi-gigahertz speeds. According to some embodiments, transistor M1may switch more than 1 amp for an electrical pulse duration betweenabout 50 ps and about 2 ns at a repetition rate between 30 Hz andapproximately 200 MHz. An example of transistor M1 includes modelATF-50189-BLK available from Avago Technologies of San Jose, Calif.Biasing and filtering circuit elements (e.g., resistors R₄, R₇, and C₃)may be connected between capacitor C₂ and the gate of transistor M1. Thedrain of transistor M1 may be directly connected to a cathode of a laserdiode or light-emitting diode 2-423, and a source of transistor M1 mayconnect to a reference potential (e.g., ground). The anode of diode2-423 may connect to a diode voltage source V_(LD). A resistor R₆ andcapacitor C₄ may be connected in parallel across diode 2-423. Accordingto some embodiments, resistor R₆ may have a value between approximately50 ohms and approximately 200 ohms, and C₄ may have a capacitancebetween approximately 5 pF and approximately 50 pF. A capacitor C₅(having a value between approximately 1 μF and approximately 5 μF) mayalso be connected between the diode voltage source V_(LD) and areference potential (e.g., ground) in parallel with the diode 2-423 andtransistor M1.

In some embodiments, a protection diode (not shown) may be connected ina reverse direction across the cathode and anode of the laser diode2-423. The protection diode may protect the laser diode from excessivereverse bias potential that could break down the laser diode junction.

In operation, a pulse from the pulse generator 2-480 momentarily turnson transistor M1, allowing current to be injected into the active regionof laser diode or light-emitting diode 2-423. In some implementations, alarge amount of forward current (e.g., up to four amps) flows throughtransistor M1 briefly. The forward current injects carriers into thelaser diode junction and produces a short or ultrashort pulse of opticalradiation. When transistor M1 turns off, parasitic inductances continuethe flow of current across the light-emitting diode or laser diode,building up charge on the cathode side of the diode, until it can bedissipated by the RC network connected in parallel with the laser diode.This temporary build-up of charge at the cathode provides a reverse biaspulse to the laser diode, and accelerates removal of carriers from theactive region. This accelerates termination of the optical pulse.

The inventors have found that the optical pulsing technique describedfor the embodiment of FIG. 2-4M is superior to pulsing techniques basedon differentiating square-wave pulses, because it can provide a higherand shorter current pulse that may be required to turn on a laser diode.

The inventors have assembled various pulse driving circuits and haveused them to drive laser diodes. FIG. 2-5A depicts another embodiment ofan assembled pulser circuit 2-500. This embodiment implements a pulser2-400 as depicted in FIG. 2-4A. In the assembled circuit, thetransmission line 2-410 is formed as a parallel-plate strip linepatterned in a U-shaped configuration on a printed circuit board, asdepicted in the figure. A GaN pHEMT transistor was used as a shuntingswitch M1 to short two ends of the U-shaped transmission line. Thepulser circuit 2-500 can be operated at repetition rates of up to 100MHz and used to drive a 50 ohm load. In some embodiments, a pulsercircuit may be operated at repetition rates between approximately 10 MHzand approximately 1 GHz.

A measured waveform from the pulser 2-500 is depicted in FIG. 2-5B. Thewaveform shows a positive pulse having an amplitude of approximately19.5 V followed by a negative pulse that reaches an amplitude ofapproximately −5 V following the positive pulse. The duration of thepositive pulse is approximately 1.5 nanoseconds. Referring again to FIG.2-4A, the pulser 2-500 was constructed to a have a terminating resistorZ_(term) of approximately 50 ohms and a pull-up or charging resistorR_(c)h of approximately 200 ohms. The value of Z_(term) was chosen toreduce power reflections from the terminating resistance back into thetransmission line. The bias applied to the transmission line 2-410 was100 V, and the switch M1 was driven at a repetition rate of 100 MHz.Approximately −1.3 V of DC bias was coupled to the diode via a bias tee,to tune the relative offset from 0 V bias. The driving pulse for theswitch M1 was a square-wave signal oscillating between approximately 0 Vand approximately 2 V.

A commercial test-bed driver was used to drive a commercial laser diode(Ushio model HL63133DG) to produce sub-100-ps optical pulses. Opticalpulse measurements are shown in FIG. 2-5C and FIG. 2-5D. As shown inFIG. 2-5C, pulses with reduced tail emission were produced at arepetition rate of 100 MHz. The average power from the laser diode wasmeasured to be about 8.3 milliwatts. The pulse duration, shown in FIG.2-5D, was measured to be approximately 84 picoseconds. The intensity ofthe optical emission from the laser diode was found to be reduced byapproximately 24.3 dB approximately 250 ps after the peak of the pulse.Even though the laser diode had a single bond wire to the diode,sub-100-ps pulses were produced. Shorter pulses (e.g., between about 25ps and about 75 ps) may be produced with multiple bond wires or withfurther improvements to the pulser circuit.

FIG. 2-6A depicts one example of a semiconductor laser 2-600 that may beused to produce optical pulses by gain switching, according to any ofthe above-described gain-switching apparatus and techniques. The laserand pulse driving circuitry may be mass produced and manufactured atlow-cost. For example, the laser may be microfabricated as anedge-emitting device using planar integrated circuit technology. Such alaser may be referred to as a slab-coupled optical waveguide laser(SCOWL). The drawing depicts an end-on, elevation view of the laser. Thelaser may be formed from a GaAs/AlGaAs material system (e.g., to emitradiation in the green, red, or infrared regions of the opticalspectrum), but other material systems (such as GaN/AlGaN) may be used insome implementations (e.g., to emit radiation in the green, blue, orultraviolet regions of the spectrum). Laser diodes may be manufacturedfrom other semiconductor material systems that include, but are notlimited to: InP, AlInGaP, InGaP, and InGaN.

According to some embodiments, a SCOWL may be formed on an n-typesubstrate or buffer layer 2-627 (e.g., a GaAs substrate or GaAs layerthat comprises Al). For example, a buffer layer may compriseAl_(x)Ga_(1-x)As where x is between approximately 0.25 and approximately0.30. The refractive index of the substrate or base layer may have afirst value n₁ that is between about 3.4 and 3.5, according to someembodiments. An electron-transport layer 2-617 of low-doped n-typesemiconductor material may be formed on the substrate 2-627. In someembodiments, the electron-transport layer 2-617 may be formed byepitaxial growth to comprise Al_(x)Ga_(1-x)As where x is betweenapproximately 0.20 and approximately 0.25 and have an n-type dopantconcentration of approximately 5×10¹⁶ cm⁻³. The thickness h of theelectron-transport layer may be between about 1 micron and about 2microns. The transport layer 2-617 may have a second value of refractiveindex n₂ that is greater than n₁. A multiple quantum well region 2-620may then be formed on the electron-transport layer 2-617. The multiplequantum well region may comprise alternating layers of materials (e.g.,alternating layers of AlGaAs/GaAs) having different dopingconcentrations that modulate energy bandgaps in the MQW region. Thelayers in the quantum well region 2-620 (which may have thicknessesbetween approximately 20 nm and approximately 200 nm) may be depositedby epitaxy, atomic layer deposition, or a suitable vapor depositionprocess. The multiple quantum well region may have an effective thirdvalue of refractive index n₃ that is greater than n₂. A hole-transportlayer 2-615 of p-type doped material may be formed adjacent the quantumwell region, and have a value of refractive index n₄ that is less thann₂. In some embodiments, the values of refractive index for thedifferent regions of a SCOWL may be as illustrated in FIG. 2-6B,according to some embodiments. In some embodiments, a SCOWL may compriseGaN semiconductor and its alloys or InP semiconductor and its alloys.

The term “adjacent” may refer to two elements arranged within closeproximity to one another (e.g., within a distance that is less thanabout one-fifth of a transverse or vertical dimension of a larger of thetwo elements). In some cases there may be intervening structures orlayers between adjacent elements. In some cases adjacent elements may beimmediately adjacent to one another with no intervening structures orelements.

After the layers of the laser device have been deposited, trenches 2-607may be etched into the layers to form an active region of the laserhaving a width w that is between about 0.25 micron and about 1.5microns. An n-contact 2-630 may be formed on a first surface of thedevice, and a p-contact 2-610 may be formed on the p-type transportlayer 2-615, adjacent the active region. Exposed surfaces of thesemiconductor layers may be passivated with an oxide or otherelectrically insulating layer, according to some embodiments.

The trenches 2-607 adjacent the active region, and the values ofrefractive indices n₁, n₂, n₃, and n₄ confine the optical mode of thelaser to a lasing region 2-625 that is adjacent to the quantum wells andunder the devices central rib, as depicted in the drawing. A SCOWL maybe designed to couple higher-order transverse modes, that mightotherwise form and lase in the lasing region 2-625, to lossyhigher-order slab modes in adjacent regions. When designed properly, allhigher-order transverse modes from the lasing region 2-625 have highrelative loss compared to the fundamental mode in the lasing region andwill not lase. In some implementations, the transverse optical mode ofthe SCOWL 2-600 may be a single transverse mode. The width of theoptical mode may be between approximately 0.5 micron and approximately 6microns. A mode profile 2-622, taken in the x direction, may be shapedas depicted in FIG. 2-6B, according to some embodiments. In otherimplementations, a SCOWL may produce multiple optical transverse modesto illuminate a region of interest. The length of the active region(along a dimension into the page) may be between 20 microns and 10 mm,in some embodiments. The output power of the SCOWL may be increased byselecting a longer length of the active region. In some embodiments, aSCOWL may deliver an average output power of more than 300 mW.

Although a semiconductor laser (e.g., a SCOWL) and pulser circuitry maybe combined to make a low-cost, ultrafast, pulsed laser suitable formany applications, the turn-off rate shown in FIG. 2-5D may not besuitable for some fluorescent lifetime analyses. In some cases, a morerapid turn-off may be needed. For example, the inventors have found thatsome measurements based on fluorescent lifetime may require the tail ofthe pulse to extinguish to a level between approximately 25 dB andapproximately 40 dB below the pulse peak within 250 ps after the pulsepeak. In some cases, the pulse power may need to drop to this range ofvalues within 100 ps after the pulse peak. In some implementations, thepulse tail may need to drop to a level between approximately 40 dB andapproximately 80 dB below the pulse peak within 250 ps after the pulsepeak. In some implementations, the pulse tail may need to drop to alevel between approximately 80 dB and approximately 120 dB below thepulse peak within 250 ps after the pulse peak.

One approach for further suppressing the emission tail of a pulse is toinclude a saturable absorber with a pulsed laser or high-brightness LEDsystem. According to some embodiments, a semiconductor saturableabsorber 2-665 may be incorporated onto a same substrate as asemiconductor laser 2-600 or high-brightness LED, as depicted in FIG.2-6C. The semiconductor laser may comprise a SCOWL structure thatincludes a quantum well region 2-620, according to some embodiments. TheSCOWL may be driven with a pulsed source 2-670, such as a pulser circuit2-400 or other pulsing circuit described above.

Adjacent to one end of the SCOWL, a saturable absorber 2-665 may beformed. The saturable absorber 2-665 may comprise a region having aband-gap that is tailored to absorb photons from the semiconductorlaser. For example, the saturable absorber may comprise a single quantumwell or multiple quantum wells that have at least one energy band gapthat is approximately equal to a characteristic energy of the laser'soptical emission. In some embodiments, a saturable absorber may beformed by ion implanting a region of the diode laser, so as toelectrically isolate the region within the diode laser cavity. Anegative bias may be applied to the region to encourage absorptionrather than gain for the same laser diode structure. At high fluencefrom the laser 2-600, the valence band of the saturable absorber maybecome depleted of carriers and the conduction band may fill, impedingfurther absorption by the saturable absorber. As a result, the saturableabsorber bleaches, and the amount of radiation absorbed from the laseris reduced. In this manner, the peak of a laser pulse may “punchthrough” the saturable absorber with a smaller attenuation in intensitythan the tail or wings of the pulse. The tail of the pulse may then besuppressed further with respect to the peak of the pulse.

According to some embodiments, a high reflector (not shown) may beformed or located at one end of the device. For example, the highreflector may be located at one end of the laser, farthest from thesaturable absorber, so as to redirect laser emission through thesaturable absorber and increase output power. According to someembodiments, an anti-reflection coating may be applied to an end of thesaturable absorber and/or SCOWL to increase extraction from the device.

According to some embodiments, a saturable absorber may include abiasing supply 2-660. The biasing supply may be used to sweep carriersout of the active region after each pulse and improve the response ofthe saturable absorber. In some embodiments, the bias may be modulated(e.g., at the pulse repetition rate) to make the saturable recovery timebe time-dependent. This modulation may further improve pulsecharacteristics. For example, a saturable absorber can suppress a pulsetail by differentially higher absorption at low intensity, if therecovery time of the saturable absorber is sufficient. Such differentialabsorption can also reduce the pulse length. The recovery time of asaturable absorber may be adjusted by applying or increasing a reversebias to the saturable absorber.

III. System Timing and Synchronization

Referring again to FIG. 1-1, regardless of the method and apparatus thatis used to produce short or ultrashort-pulses, a system 1-100 mayinclude circuitry configured to synchronize at least some electronicoperations (e.g., data acquisition and signal processing) of an analyticsystem 1-160 with the repetition rate of optical pulses from the opticalsource. There are at least two ways to synchronize the pulse repetitionrate to electronics on the analytic system 1-160. According to a firsttechnique, a master clock may be used as a timing source to trigger bothgeneration of pulses at the pulsed optical source and instrumentelectronics. In a second technique, a timing signal may be derived fromthe pulsed optical source and used to trigger instrument electronics.

FIG. 3-1 depicts a system in which a clock 3-110 provides a timingsignal at a synchronizing frequency f_(sync) to both a pulsed opticalsource 1-110 (e.g., a gain-switched pulsed laser or pulsed LED) and toan analytic system 1-160 that may be configured to detect and processsignals that result from interactions between each excitation pulse1-120 and biological, chemical, or other physical matter. As just oneexample, each excitation pulse may excite one or more fluorescentmolecules of a biological sample that are used to analyze a property ofthe biological sample (e.g., cancerous or non-cancerous, viral orbacterial infection, blood glucose level). For example, non-cancerouscells may exhibit a characteristic fluorescent lifetime of a first valueτ₁, whereas cancerous cells may exhibit a lifetime of a second value τ₂that is different from and can be distinguished from the first lifetimevalue. As another example, a fluorescent lifetime detected from a sampleof blood may have a lifetime value and/or intensity value (relative toanother stable marker) that is dependent on blood glucose level. Aftereach pulse or a sequence of several pulses, the analytic system 1-160may detect and process fluorescent signals to determine a property ofthe sample. In some embodiments, the analytic system may produce animage of an area probed by the excitation pulses that comprises a two orthree-dimensional map of the area indicating one or more properties ofregions within the imaged area.

Regardless of the type of analysis being done, detection and processingelectronics on the analytic system 1-160 may need to be carefullysynchronized with the arrival of each optical excitation pulse. Forexample, when evaluating fluorescent lifetime, it is beneficial to knowthe time of excitation of a sample accurately, so that timing ofemission events can be correctly recorded.

A synchronizing arrangement depicted in FIG. 3-1 may be suitable forsystems in which the optical pulses are produced by active methods(e.g., external control). Active pulsed systems may include, but are notlimited to gain-switched lasers and pulsed LEDs. In such systems, aclock 3-110 may provide a digital clock signal that is used to triggerpulse production (e.g., gain switching or current injection into an LEDjunction) in the pulsed optical source 1-110. The same clock may alsoprovide the same or synchronized digital signal to an analytic system1-160, so that electronic operations on the instrument can besynchronized to the pulse-arrival times at the instrument.

The clock 3-110 may be any suitable clocking device. In someembodiments, the clock may comprise a crystal oscillator or a MEMS-basedoscillator. In some implementations, the clock may comprise a transistorring oscillator.

The frequency f_(sync) of a clock signal provided by the clock 3-110need not be a same frequency as the pulse repetition rate R. The pulserepetition rate may be given by R=1/T, where T is the pulse-separationinterval. In FIG. 3-1, the optical pulses 1-120 are depicted as beingspatially separated by a distance D. This separation distancecorresponds to the time T between arrival of pulses at the analyticsystem 1-160 according to the relation T=D/c where c is the speed oflight. In practice, the time T between pulses can be determined with aphotodiode and oscilloscope. According to some embodiments, T=f_(sync)/Nwhere N is an integer greater than or equal to 1. In someimplementations, T=Nf_(sync) where N is an integer greater than or equalto 1.

FIG. 3-2 depicts a system in which a timer 3-220 provides asynchronizing signal to the analytic system 1-160. In some embodiments,the timer 3-220 may derive a synchronizing signal from the pulsedoptical source 1-110, and the derived signal is used to provide asynchronizing signal to the analytic system 1-160.

According to some embodiments, the timer 3-220 may receive an analog ordigitized signal from a photodiode that detects optical pulses from thepulse source 1-110. The timer 3-220 may use any suitable method to formor trigger a synchronizing signal from the received analog or digitizedsignal. For example, the timer may use a Schmitt trigger or comparatorto form a train of digital pulses from detected optical pulses. In someimplementations, the timer 3-220 may further use a delay-locked loop orphase-locked loop to synchronize a stable clock signal to a train ofdigital pulses produced from the detected optical pulses. The train ofdigital pulses or the locked stable clock signal may be provided to theanalytic system 1-160 to synchronize electronics on the instrument withthe optical pulses.

In some embodiments, two or more pulsed optical sources 1-110 a, 1-110 bmay be needed to supply optical pulses at two or more differentwavelengths to an analytic system 1-160, as depicted in FIG. 3-3. Insuch embodiments, it may be necessary to synchronize pulse repetitionrates of the optical sources and electronic operations on the analyticsystem 1-160. In some implementations, if two pulsed optical sources useactive methods to produce pulses, the techniques described above inconnection with FIG. 3-1 may be used. For example, a clock 3-110 maysupply a clock or synchronizing signal at a synchronizing frequencyf_(sync) to both pulsed optical sources 1-110 a, 1-110 b, and to theanalytic system 1-160.

In some implementations, it may be beneficial to interleave pulses intime from two pulsed optical sources, as depicted in FIG. 3-4A and FIG.3-4B. When pulses are interleaved, a pulse 3-120 a from a first source1-110 a may excite one or more samples at the analytic system 1-160 witha first characteristic wavelength λ₁ at a first time t₁. Datarepresentative of the first pulse's interaction with the one or moresamples may then be collected by the instrument. At a later time t₂, apulse 3-120 b from a second source 1-110 b may excite one or moresamples at the analytic system 1-160 with a second characteristicwavelength λ₂. Data representative of the second pulse's interactionwith the one or more samples may then be collected by the instrument. Byinterleaving the pulses, effects of pulse-sample interactions at onewavelength may not intermix with effects of pulse-sample interactions ata second wavelength. Further, characteristics associated with two ormore fluorescent markers may be detected.

Pulses may be interleaved with timing and synchronization circuitry, asdepicted in FIG. 3-4A. Methods described in connection with FIG. 3-3 maybe used to synchronize pulse trains from the two pulsed optical sources1-110 a, 1-110 b, and to synchronize electronics and operations on theanalytic system 1-160 with the arrival of pulses. To interleave thepulses, pulses of one pulsed optical source may be phase-locked ortriggered out of phase with pulses from the other pulsed optical source.For example, pulses of a first pulsed optical source 1-110 a may bephase-locked (using a phase-locked loop or delay-locked loop) ortriggered to be 180 degrees out of phase with pulses from the secondpulsed optical source 1-110 b, though other phase or angle relationshipsmay be used in some embodiments. In some implementations, a timing delaymay be added to a trigger signal provided to one of the pulsed opticalsources. The timing delay may delay a trigger edge by approximatelyone-half the pulse-separation interval T. According to some embodiments,a frequency-doubled synchronization signal may be generated by a timer3-220, and provided to the instrument 3-160 for synchronizing instrumentelectronics and operations with the arrival of interleaved pulses fromthe pulsed optical sources.

IV. Time-Domain Applications for Pulsed Optical Sources

Pulsed optical sources described above are useful for varioustime-domain applications. In some embodiments, pulsed optical sourcesmay be used in systems configured to detect and/or characterize acondition or property of a biological sample based on fluorescentlifetimes, fluorescent wavelengths, fluorescent intensities, or acombination thereof. Pulsed optical sources may also be used intime-of-flight systems. Time-of-flight systems may include imagingsystems and ranging systems that illuminate a target with a short orultrashort optical pulse, and then detect backscattered radiation fromthe target to form a three-dimensional image of the target or determinea distance to the target.

In a time-domain application that utilizes fluorescence, a pulsedoptical source operating at a first characteristic wavelength may exciteone or more fluorescent molecules in a sample, and the analytic systemmay detect and analyze fluorescent emission from the sample at one ormore wavelengths that are different from the pulsed optical source'swavelength. According to some embodiments, one or more properties of abiological sample may be determined based upon an analysis offluorescent lifetimes from one or more fluorescent molecules present inthe sample. In some implementations, additional characteristics offluorescent emission (e.g., wavelength, intensity) may be analyzed tofurther aid in determination of one or more properties of a biologicalsample. Systems that determine properties of biological samples based onfluorescent lifetimes may be imaging or non-imaging systems. Whenconfigured as an imaging system, a pixel array may be used forfluorescent detection, and imaging optics may be placed between thesample and pixel array to form an image of at least a portion of thesample on the pixel array. In some implementations, a non-imaging systemmay use a pixel array to detect fluorescence from a plurality of samplesin parallel.

An instrument 4-100 for determining properties of biological samplesbased at least in part on fluorescent lifetime analysis and using pulsedoptical sources is depicted in FIG. 4-1, according to some embodiments.Such an instrument may comprise one or more pulsed optical sources4-120, a time-binning photodetector 4-150, an optical system 4-130(which may be one or more lenses, and may include one or more opticalfilters), and a transparent window 4-140 that may be pressed against asubject or on which a biological sample may be placed. The pulsedoptical source or sources and optical system may be arranged so thatoptical pulses from the source or sources illuminate an area through thewindow 4-140. Fluorescent emission that is excited by the opticalexcitation pulses may be collected by the optical system 4-130 anddirected to the time-binning photodetector 4-150 which may discernlifetimes of one or more fluorescent molecules, as described furtherbelow. In some implementations, photodetector 4-150 may be non-imaging.In some implementations, photodetector 4-150 may comprise an array ofpixels, each having time-binning capability, to form images of a sample.The image data may include spatially-resolved fluorescent lifetimeinformation as well as conventional imaging information. Components ofthe instrument may be mounted in a casing 4-105, which may be small insize so that the instrument can be operated as a hand-held device. Theoptical source(s) 4-120 and photodetector 4-150 may or may not bemounted on a same circuit board 4-110. In some embodiments, theinstrument 4-100 may include a microprocessor or microcontroller, and/ormay include data-communication hardware so that data can be transmittedto an external device (e.g., a smart phone, laptop, PC) for processingand/or data storage.

Systems configured to analyze samples based on fluorescent lifetimes maydetect differences in fluorescent lifetimes between differentfluorescent molecules, and/or differences between lifetimes of the samefluorescent molecules in different environments that affect fluorescentlifetimes. By way of explanation, FIG. 4-2 plots two differentfluorescent emission probability curves (A and B), which may berepresentative of fluorescent emission from two different fluorescentmolecules, for example, or a same fluorescent molecule in differentenvironments. With reference to curve A, after being excited by a shortor ultrashort optical pulse, a probability p_(A)(t) of a fluorescentemission from a first molecule may decay with time, as depicted. In somecases, the decrease in the probability of a photon being emitted overtime may be represented by an exponential decay functionp_(A)(t)=P_(Ao)e^(−t/τ) ^(A) , where P_(Ao) is an initial emissionprobability and τ_(A) is a temporal parameter associated with the firstfluorescent molecule that characterizes the emission decay probability.τ_(A) may be referred to as the “fluorescence lifetime,” “emissionlifetime,” or “lifetime” of the first fluorescent molecule. In somecases, the value of τ_(A) may be altered by a local environment of thefluorescent molecule. Other fluorescent molecules may have differentemission characteristics than that shown in curve A. For example,another fluorescent molecule may have a decay profile that differs froma single exponential decay, and its lifetime may be characterized by ahalf-life value or some other metric.

A second fluorescent molecule may have a decay profile that isexponential, but has a measurably different lifetime τ_(B), as depictedfor curve B in FIG. 4-2. Different fluorescent molecules may havelifetimes or half-life values ranging from about 0.1 ns to about 20 ns,in some embodiments. In the example shown, the lifetime for the secondfluorescent molecule of curve B is shorter than the lifetime for curveA, and the probability of emission is higher sooner after excitation ofthe second molecule than for curve A.

The inventors have recognized and appreciated that differences influorescent emission lifetimes can be used to discern between thepresence or absence of different fluorescent molecules and/or to discernbetween different environments or conditions in a sample that affectlifetime of a fluorescent molecule or molecules. In some cases,discerning fluorescent molecules based on lifetime (rather than emissionwavelength, for example) can simplify some aspects of an analytic system1-160. As an example, wavelength-discriminating optics (such aswavelength filters, dedicated detectors for each wavelength, dedicatedpulsed optical sources at different wavelengths, and/or diffractiveoptics) may be reduced in number or eliminated when discerningfluorescent molecules based on lifetime. In some cases, a single pulsedoptical source may be used to excite different fluorescent moleculesthat emit within a same wavelength region of the optical spectrum buthave measurably different lifetimes. An analytic system that uses asingle pulsed optical source, rather than multiple sources at differentwavelengths, to excite and discern different fluorescent moleculesemitting in a same wavelength region can be less complex to operate andmaintain, more compact, and may be manufactured at lower cost.

Although analytic systems based on fluorescent lifetime analysis mayhave certain benefits, the amount of information obtained by an analyticsystem may be increased by allowing for additional detection techniques.For example, some analytic systems 1-160 may additionally be configuredto discern one or more properties of a sample based on fluorescentwavelength and/or fluorescent intensity.

Referring again to FIG. 4-2, according to some embodiments, differentfluorescent lifetimes may be distinguished with a photodetector that isconfigured to time-bin fluorescent emission events following excitationof a fluorescent molecule. The time binning may occur during a singlecharge-accumulation cycle for the photodetector. The concept ofdetermining fluorescent lifetime by time-binning of emission events isdepicted graphically in FIG. 4-3. At time t₁ or just prior to t₁, afluorescent molecule or ensemble of fluorescent molecules of a same type(e.g., the type corresponding to curve B of FIG. 4-2) is (are) excitedby a short or ultrashort optical pulse. For an ensemble of molecules,the intensity of emission may have a time profile as depicted in FIG.4-3.

For a single molecule or a small number of molecules, however, theemission of fluorescent photons occurs according to the statistics ofcurve B in FIG. 4-2. A time-binning photodetector 4-150 may accumulateemission events into discrete time bins (three indicated in FIG. 4-3)that are measured with respect to the excitation time of the fluorescentmolecule(s). When a large number of emission events are summed, theresulting time bins may approximate the decaying intensity curve shownin FIG. 4-3, and the binned signals can be used to distinguish betweendifferent fluorescent molecules or different environments in which afluorescent molecule is located.

Examples of a time-binning photodetector are described in internationalapplication No. PCT/US2015/044360, which is incorporated herein byreference, and an embodiment of such a photodetector for explanationpurposes is depicted in FIG. 4-4. A single time-binning photodetector4-400 may comprise a photon-absorption/carrier-generation region 4-402,a carrier-travel region 4-406, and a plurality of carrier-storage bins4-408 a, 4-408 b, 4-408 c all formed on a semiconductor substrate. Thecarrier-travel region may be connected to the plurality ofcarrier-storage bins by carrier-transport channels 4-407. Only threecarrier-storage bins are shown, but there may be more. There may be aread-out channel 4-410 connected to the carrier-storage bins. Thephoton-absorption/carrier-generation region 4-402, carrier-travel region4-406, carrier-storage bins 4-408 a, 4-408 b, 4-408 c, and read-outchannel 4-410 may be formed by doping the semiconductor locally and/orforming adjacent insulating regions to provide photodetection capabilityand confine carriers. A time-binning photodetector 4-400 may alsoinclude a plurality of electrodes 4-420, 4-422, 4-432, 4-434, 4-436,4-440 formed on the substrate that are configured to generate electricfields in the device for transporting carriers through the device.

In operation, fluorescent photons may be received at thephoton-absorption/carrier-generation region 4-402 at different times andgenerate carriers. For example, at approximately time t₁ threefluorescent photons may generate three carrier electrons in a depletionregion of the photon-absorption/carrier-generation region 4-402. Anelectric field in the device (due to doping and/or an externally appliedbias to electrodes 4-420 and 4-422, and optionally or alternatively to4-432, 4-434, 4-436) may move the carriers to the carrier-travel region4-406. In the carrier-travel region, distance of travel translates to atime after excitation of the fluorescent molecules. At a later time t₅,another fluorescent photon may be received in thephoton-absorption/carrier-generation region 4-402 and generate anadditional carrier. At this time, the first three carriers have traveledto a position in the carrier-travel region 4-406 adjacent to the secondstorage bin 4-408 b. At a later time t₇, an electrical bias may beapplied between electrodes 4-432, 4-434, 4-436 and electrode 4-440 tolaterally transport carriers from the carrier-travel region 4-406 to thestorage bins. The first three carriers may then be transported to andretained in the first bin 4-408 a and the later-generated carrier may betransported to and retained in the third bin 4-408 c. In someimplementations, the time intervals corresponding to each storage binare at the sub-nanosecond time scale, though longer time scales may beused in some embodiments (e.g., in embodiments where fluorophores havelonger decay times).

The process of generating and time-binning carriers after an excitationevent (e.g., excitation pulse from a pulsed optical source) may occuronce after a single excitation pulse or be repeated multiple times aftermultiple excitation pulses during a single charge-accumulation cycle forthe photodetector 4-400. After charge accumulation is complete, carriersmay be read out of the storage bins via the read-out channel 4-410. Forexample, an appropriate biasing sequence may be applied to at leastelectrode 4-440 and a downstream electrode (not shown) to removecarriers from the storage bins 4-408 a, 4-408 b, 4-408 c.

Aspects of signal acquisition are depicted in further detail formultiple excitation pulses in FIG. 4-5A and FIG. 4-5B. In FIG. 4-5A,multiple excitation pulses are applied to a sample at times t_(e1),t_(e2), t_(e3), . . . . Following each excitation pulse, one or morefluorescent emission events may occur at times t_(fn), which lead to theaccumulation of carriers into the different carrier-storage binsdepending on when the emission event occurs. After a number ofexcitation events, the accumulated signal in each carrier-storage binmay be read out to provide a signal sequence, which may be representedas a histogram 4-510 (depicted in FIG. 4-5B). The signal sequence mayindicate a number of photons detected during each binned time intervalafter excitation of the fluorophore(s) in a sample, and isrepresentative of a fluorescent emission decay rate. The signalsequence, or histogram, may be used to distinguish between differentfluorescent molecules or different environments in which a fluorescentmolecule exists.

As an example of distinguishing different fluorescent molecules, aphotodetector having three time bins, as depicted in FIG. 4-3B and FIG.4-4, may produce three signal values (35, 9, 3.5), which are representedby the histogram for bin1-bin3 of FIG. 4-5B and correspond to curve B inFIG. 4-2. These binned signal values may have different relative and/orabsolute values than binned signal values recorded from a differentfluorescent molecule, such as one corresponding to curve A in FIG. 4-2,which might produce the binned values (18, 12, 8). By comparing thesignal sequence of binned values against a calibration standard, it ispossible to distinguish between two or more fluorescent molecules orenvironments that affect fluorescent lifetime. It can be beneficial thatmultiple different fluorescent molecules and/or environments may bedistinguished based on lifetime information using a pulsed opticalsource operating at only a single characteristic wavelength.

According to some embodiments, an excitation bin (e.g., bin 0) may beincluded in at least one time-binning photodetector to record a signallevel for the excitation pulse (e.g., accumulate carriers directlygenerated by the excitation pulse). The recorded signal level may beused to normalize fluorescent signal levels, which can be useful fordistinguishing fluorescent molecules based on intensity.

In some embodiments, the signal values from the storage bins 4-408 maybe used to fit an emission decay curve (e.g., a single exponentialdecay) and determine a detected lifetime. In some embodiments, thebinned signal values may be fit to multiple exponential decays, such asdouble or triple exponentials. A Laguerre decomposition process may beused to analyze multiple exponential decays. In some implementations,the signal values may be treated as a vector or location and mapped toM-dimensional space, and cluster analysis may be used to determine adetected lifetime. Once a lifetime has been determined, the type offluorescent molecule or a property of the environment in which afluorescent molecule is located may be identified.

Although the example described in connection with FIG. 4-3 and FIG. 4-4depicts three time bins, a time-binning photodetector may have fewer ormore time bins. For example, the number of time bins may be 2, 3, 4, 5,6, 7, 8, or more. In some cases, there may be 16, 32, 64, or more timebins. According to some embodiments, a number of time bins in aphotodetector may be reconfigurable. For example, one or more adjacentbins may be combined when read-out.

Although the discussion for FIG. 4-3 relates to detecting emission froma single type of fluorescent molecule at a time, in some cases a samplemay contain two or more different fluorescent molecules having differentlifetimes. Where multiple different fluorescent molecules contribute toa temporal emission profile, an average fluorescence lifetime may beused to represent the ensemble. In some embodiments, an analytic system1-160 may be configured to discern between combinations of fluorescentmolecules. For example, a first combination of fluorescent molecules mayexhibit a different average lifetime than a second combination offluorescent molecules.

According to some embodiments, time-binning photodetectors may be usedin an imaging array, and imaging optics may be included between thetime-binning photodetector array and a sample. For example, each imagingpixel of an imaging array may comprise a time-binning photodetector4-400. The imaging optics may form an image of a region of the sample onthe photodetector array. Each pixel in the photodetector array mayrecord time-binned signal values that are analyzed to determine afluorescent lifetime for the portion of the imaged region correspondingto the pixel. Accordingly, such an imaging array can provide spatiallyresolved fluorescent lifetime imaging information to discern differentregions in an image having different fluorescent lifetimecharacteristics. In some implementations, the same time-binningphotodetectors may be used to obtain a conventional image of the sameregion, e.g., by summing all bins at each pixel or by constructing animage from the excitation pulse bin (bin0). Fluorescent lifetimevariations may be displayed as an overlapping color-coded map on aconventional gray-scale or color image. In some cases, the lifetimemapping may enable a physician performing a procedure to identify anabnormal or diseased region of tissue (e.g., cancerous orpre-cancerous).

The inventors have recognized and appreciated that compact, pulsedoptical sources and time-binning photodetectors for detectingfluorescent lifetimes may be combined in low-cost, portable,point-of-care (POC) instruments that can have applications in clinicalsettings or at-home settings. Such instruments may be imaging ornon-imaging, and may utilize fluorescent lifetime analysis to determineone or more properties of a biological sample (e.g., human tissue). Insome cases, an instrument 4-100 for determining properties of biologicalsamples may be used in the field for analyzing biological substances(e.g., for analyzing potentially hazardous material). Some aspects ofPOC instruments and sample analysis using fluorescent lifetimes aredescribed below.

The inventors have recognized and appreciated that some endogenousbiological molecules fluoresce with signature lifetimes that may beanalyzed to determine a patient's condition or a condition of apatient's tissue or organ. Accordingly, some native biological moleculesmay serve as endogenous fluorescent molecules for a region of a patient,and provide label-free reporters for that region of the patient.Examples of endogenous fluorescent molecules may include hemoglobin,collagen, nicotinamide adenine dinucleotide phosphate (NAD(P)H),retinol, riboflavin, cholecalciferol, folic acid, pyridoxine, tyrosine,dityrosine, glycation adduct, idolamine, lipofuscin, polyphenol,tryptophan, flavin, and melanin, by way of example and not limitation.

Endogenous fluorescent molecules may vary in the wavelength of lightthey emit and their response to excitation energy. Wavelengths ofexcitation and fluorescence for some exemplary endogenous fluorescentmolecules are provided in Table 1. Additional endogenous fluorescentmolecules and their characteristic fluorescent wavelengths include:retinol—500 nm, riboflavin—550 nm, cholecalciferol—380-460 nm, andpyridoxine—400 nm.

TABLE 1 Endogenous Fluorescent Molecules Molecule Excitation (nm)Fluorescence (nm) NAD(P)H 340 450 Collagen 270-370 305-450 Tyrosine 270305 Dityrosine 325 400 Excimer-like aggregate 270 360 Glycation adduct370 450 Tryptophan 280 300-350 Falvin 380-490 520-560 Melanin 340-400360-560

Endogenous fluorescent molecules may also have different fluorescentlifetimes and/or fluorescent lifetimes that are sensitive to asurrounding environment. Environmental factors that may affectfluorescent lifetimes of endogenous fluorescent molecules include,changes in tissue architecture, morphology, oxygenation, pH,vascularity, cell structure and/or cell metabolic state. In someembodiment, a fluorescent lifetime (or average of combined lifetimes)for a healthy tissue may be different than for an unhealthy tissue.Analyzing fluorescent lifetimes detected from a patient's tissue thathas been illuminated with a short or ultrashort optical pulse may allowa clinician to detect an earlier stage of a disease in the patient thanother assessment techniques. For example, some types of skin cancer maybe detected at an early stage using fluorescent lifetime analysis beforethe cancer is visible to the unaided eye.

In some embodiments, the presence and/or relative concentrations ofcertain biological molecules may be detected to determine a patient'scondition. For some biological molecules, the oxidation state of themolecule may provide an indication of the patient's condition. Afluorescent lifetime for the molecule may be altered based upon anoxidation state of the molecule. Analysis of detected fluorescentlifetimes may be used to determine the relative concentrations of anoxidized state and a reduced state of a biological molecule in thetissue of a patient. The relative concentrations may indicate acondition of the patient. In some cases, some biological molecules(e.g., NADH) may bind to other molecules (e.g., proteins) in a cell aswell as have an unbound or free solution state. The bound and unboundstates may have different fluorescent lifetimes. Assessment of a cell ortissue may include determining relative concentrations of molecules infree versus bound forms based upon fluorescent lifetimes.

Certain biological molecules may provide an indication of a variety ofdiseases and conditions including cancer (e.g., melanoma), tumors,bacterial infection, virial infection, and diabetes. As an example,cancerous cells and tissues may be differentiated from healthy cells andtissues by analyzing fluorescent lifetimes from certain biologicalmolecules (e.g., NAD(P)H, riboflavin, flavin). A cancerous tissue mayhave a higher concentration of one or more of these biological moleculesthan a healthy tissue. As another example, diabetes in individuals maybe assessed by detecting fluorescent lifetimes associated withbiological molecules that are indicative of glucose concentration, suchas hexokinase and glycogen adduct. As another example, general changesdue to aging may be assessed by detecting concentrations of collagen andlipofuscin based on fluorescent lifetimes.

In some embodiments, exogenous fluorescent molecules may be incorporatedinto a region of tissue, and be used alternatively, or in addition to,endogenous fluorescent molecules. In some cases, exogenous fluorescentmarkers may be included with a probe or provided as a marker to identifythe presence of a target (e.g., a particular molecule, bacteria, orvirus) in the sample. Examples of exogenous fluorescent moleculesinclude fluorescent stains, organic dyes, fluorescent proteins, enzymes,and/or quantum dots. Such exogenous molecules may be conjugated to aprobe or functional group (e.g., molecule, ion, and/or ligand) thatspecifically binds to a particular target or component suspected to bepresent in the sample. Attaching an exogenous fluorescent molecule to aprobe may allow the identification of the target by detecting afluorescent lifetime indicative of the exogenous fluorescent molecule.In some embodiments, exogenous fluorescent molecules may be included ina composition (e.g., gel or liquid) that can be easily applied to apatient (e.g., topical application to skin, ingestion forgastrointestinal tract imaging).

As may be appreciated, a compact, POC imaging instrument may allow aclinician to evaluate and/or diagnose a patient's condition in anon-invasive manner. By imaging an accessible region of tissue with animaging device rather than by extracting a biological sample from apatient, assessments of the patient may be performed in a manner thatreduces the amount of time involved in obtaining results, reduces theinvasiveness of a procedure, reduces the cost, and/or facilitates theability of clinicians to treat patients without moving the patient to aremote testing location or sending a sample of a patient to a testingfacility.

Another application for time-domain, fluorescent lifetime imaging is inthe area of microscopy. Fluorescence lifetime imaging microscopy (FLIM)may be performed by exciting a sample viewed microscopically with ashort or ultrashort optical pulse, and detecting the fluorescence fromthe sample with a time-binning photodetector array. The detectedfluorescence may be analyzed at the pixel level to determine lifetimesfor corresponding imaged portions within the field of view of themicroscope, and lifetime data may be mapped to a resulting image of thesample. Accordingly, sample properties may be determined at themicroscopic level based on fluorescent lifetimes.

Pulsed optical sources and time-binning photodetector arrays may also beused in time-domain applications that do not involve fluorescentlifetime analysis. One such application includes time-of-flight (TOF)imaging. In TOF imaging, optical pulses may be used to illuminate adistant object. Imaging optics may be used to collect backscatteredradiation from the pulses and form an image of the distant object on atime-binning photodetector array. At each pixel in the array, thearrival time of photons may be determined (e.g., determining when a peakof a backscattered pulse occurs). Since the arrival time is proportionalto the distance between the object and the photodetector array, athree-dimensional map of the object may be created that shows surfacetopography of the imaged object.

V. Configurations

Various configurations and embodiments of the apparatus and methods maybe implemented. Some example configurations are described in thissection, but the invention is not limited to only the listedconfigurations and embodiments.

(1) A pulsed optical source comprising a semiconductor diode configuredto emit light, and a driving circuit that includes a transistor coupledto a terminal of the semiconductor diode, wherein the driving circuit isconfigured to receive a unipolar pulse and apply a bipolar electricalpulse to the semiconductor diode responsive to receiving the unipolarpulse.

(2) The pulsed optical source of configuration (1), wherein the bipolarelectrical pulse comprises a first pulse having a first magnitude andfirst polarity that is followed by a second pulse of opposite polarityhaving a second magnitude different from the first magnitude.

(3) The pulsed optical source of (2), wherein the second magnitude isbetween 25% and 90% of the first magnitude.

(4) The pulsed optical source of any one of (1)-(3), further comprisingmultiple wire bonds connected to a terminal of the semiconductor diode.

(5) The pulsed optical source of any one of (1)-(4), further comprisinga pulse generator coupled to the driving circuit and configured to formthe unipolar pulse and output the unipolar pulse to the driving circuit.

(6) The pulsed optical source of (5), wherein the pulse generator,driving circuit, and semiconductor diode are located on a same printedcircuit board.

(7) The pulsed optical source of (5), wherein the pulse generator,driving circuit, and semiconductor diode are located on a samesubstrate.

(8) The pulsed optical source of any one of (1)-(7), wherein a pulselength of the unipolar pulse is between 50 ps and 500 ps.

(9) The pulsed optical source of any one of (5)-(8), wherein the pulsegenerator comprises a first logic gate that forms the unipolar pulsefrom two differential clock signals.

(10) The pulsed optical source of (9), wherein the first logic gatecomprises an emitter-coupled logic gate.

(11) The pulsed optical source of (9) or (10), wherein the pulsegenerator further comprises a fan-out gate configured to receive asingle clock signal and output four clock signals to the first logicgate.

(12) The pulsed optical source of any one of (9)-(11), wherein the pulsegenerator further comprises an adjustable delay element configured tovary a pulse length of the unipolar pulse in increments between 1 ps and5 ps.

(13) The pulsed optical source of any one of (9)-(12), wherein thetransistor has current-carrying terminals connected between a cathode ofthe semiconductor diode and a reference potential and has a gateterminal coupled to the first logic gate.

(14) The pulsed optical source of (13), further comprising a capacitorconnected between the gate terminal of the transistor and an output fromthe first logic gate.

(15) The pulsed optical source of any one of (1)-(14), wherein thetransistor comprises a high-electron-mobility field-effect transistor.

(16) The pulsed optical source of any one of (1)-(15), wherein thetransistor is configured to switch up to 4 amps through thesemiconductor diode for a duration between 50 ps and 2 ns.

(17) The pulsed optical source of any one of (9)-(13), furthercomprising a second logic gate connected in parallel with the firstlogic gate and arranged to form a second unipolar pulse from the twodifferential clock signals, wherein an output from the second logic gateis coupled to the gate terminal of the transistor.

(18) The pulsed optical source of any one of (1)-(17), wherein a drainterminal of the transistor connects directly to a cathode of thesemiconductor diode.

(19) The pulsed optical source of (18), further comprising a firstcapacitor and resistor connected in parallel to the drain terminal.

(20) The pulsed optical source of (18) or (19), further comprising asecond capacitor connected between an anode of the semiconductor diodeand a source terminal of the transistor.

(21) The pulsed optical source of any one of (5)-(20), wherein the pulsegenerator and driving circuit are configured to modulate thesemiconductor diode with the bipolar electrical pulse at a repetitionrate of between about 30 Hz and about 200 MHz.

(22) The pulsed optical source of any one of (1)-(21), wherein anoptical pulse having a full-width-half maximum duration between 50 psand 500 ps is emitted from the semiconductor diode responsive toapplication of the bipolar electrical pulse.

(23) The pulsed optical source of any one of (1)-(21), wherein theoptical pulse has a characteristic wavelength selected from thefollowing group: 270 nm, 280 nm, 325 nm, 340 nm, 370 nm, 380 nm, 400 nm,405 nm, 410 nm, 450 nm, 465 nm, 470 nm, 490 nm, 515 nm, 640 nm, 665 nm,808 nm, and 980 nm.

(24) The pulsed optical source of any one of (1)-(23), wherein a tail ofthe optical pulse remains below at least 20 dB from the peak of thepulse after 250 ps from the peak of the pulse.

(25) The pulsed optical source of any one of (1)-(24), wherein thesemiconductor diode comprises a laser diode.

(26) The pulsed optical source of (25), wherein the laser diode includesmultiple quantum wells.

(27) The pulsed optical source of any one of (1)-(26), wherein thesemiconductor diode is a light-emitting diode.

(28) The pulsed optical source of any one of (1)-(27), wherein thesemiconductor diode is a slab-coupled optical waveguide laser diode.

(29) The pulsed optical source of any one of (1)-(28), furthercomprising a saturable absorber arranged to receive an optical pulsefrom the semiconductor diode.

(30) The pulsed optical source of any one of (1)-(29), wherein thesaturable absorber is formed in a same substrate as the semiconductordiode.

(31) The pulsed optical source of any one of (1)-(4), (15), (16), (18),and (22)-(30), wherein the driving circuit comprises a transmission linepulse generator.

(32) The pulsed optical source of (31), further comprising atransmission line that is formed in a U shape.

(33) The pulsed optical source of claim 31) or (32), wherein thesemiconductor diode is connected to a first end of the transmission lineand further comprising a terminating impedance that is connected to asecond end of the transmission line.

(34) The pulsed optical source of claim 33), further comprising ashorting transistor that is arranged to short the first end and secondend of the transmission line to a reference potential.

(35) The pulsed optical source of any one of (1)-(34), furthercomprising a photodetector array having a plurality of pixels that areeach configured to discriminate photon arrival times into at least twotime bins during a single charge-accumulation interval, and an opticalsystem arranged to form an image of an object, that is illuminated bythe pulsed optical source, on the photodetector array.

(36) The pulsed optical source of (35), wherein the photodetector arrayis arranged to produce signals representative of fluorescent lifetime ofat least one fluorescent molecule located at the distant object.

(37) The pulsed optical source of (35) or (36), further comprisingsignal processing electronics that are configured to receive the signalsrepresentative of fluorescent lifetime from the photodetector array andgenerate digital data for an electronic image of the object, wherein theelectronic image indicates at least one characteristic of the objectbased on fluorescent lifetime.

(38) A method of producing an optical pulse, the method comprising actsof receiving at least one clock signal, producing an electrical pulsefrom the at least one clock signal, driving a gate terminal of atransistor with the electrical pulse, wherein a current carryingterminal of the transistor is connected to a semiconductor diode that isconfigured to emit light, and applying a bipolar current pulse to thesemiconductor diode to produce an optical pulse responsive to activationof the transistor by the electrical pulse.

(39) The method of embodiment (38), wherein the electrical pulse is aunipolar pulse.

(40) The method of (38) or (39), further comprising adjusting a pulseduration and not a pulse amplitude of the unipolar pulse to control anamplitude of the optical pulse.

(41) The method of any one of (38)-(40), wherein the optical pulse has afull-width-half-maximum duration between 50 ps and 2 ns.

(42) The method of any one of (38)-(40), wherein the optical pulse has afull-width-half-maximum duration between 50 ps and 500 ps.

(43) The method of any one of (38)-(42), wherein the optical pulse has acharacteristic wavelength selected from the following group: 270 nm, 280nm, 325 nm, 340 nm, 370 nm, 380 nm, 400 nm, 405 nm, 410 nm, 450 nm, 465nm, 470 nm, 490 nm, 515 nm, 640 nm, 665 nm, 808 nm, and 980 nm.

(44) The method of any one of (38)-(43), further comprising repeatingthe acts of receiving, producing, driving, and applying to produce aseries of optical pulses at a repetition rate between 30 Hz and 200 MHz.

(45) The method of any one of (38)-(44), wherein the bipolar currentpulse comprises a first pulse having a first amplitude and a secondpulse having a second amplitude of opposite polarity and differentmagnitude from the first pulse.

(46) The method of (38)-(45), wherein the semiconductor diode comprisesa laser diode or light-emitting diode.

(47) The method of any one of (38)-(46), further comprisingdifferentially attenuating a portion of the optical pulse with asaturable absorber.

(48) The method of any one of (38)-(47), wherein the act of receiving atleast one clock signal comprises receiving two differential clocksignals at a logic gate coupled to the gate terminal of the transistor.

(49) The method of any one of (38)-(47), wherein the act of receiving atleast one clock signal comprises receiving two differential clocksignals at two logic gates coupled in parallel the gate terminal of thetransistor.

(50) The method of any one of (38)-(49), wherein the act of producingthe electrical pulse comprises processing two differential clock signalswith a logic gate coupled to the gate terminal of the transistor to formthe electrical pulse.

(51) The method of (50), further comprising setting a length of theelectrical pulse by a phase delay between the two differential clocksignals.

(52) The method of any one of (38)-(51), wherein the act of producingthe electrical pulse comprises processing two differential clock signalswith two logic gates coupled in parallel to the gate terminal of thetransistor to form the electrical pulse.

(53) The method of any one of (38)-(52), further comprising illuminatinga sample with optical pulses from the semiconductor diode, and detectingfluorescent lifetimes from the sample.

(54) The method of (53), further comprising distinguishing between atleast two different fluorescent lifetimes having different decay ratesassociated with two different fluorescent molecules or environments inwhich the molecules are located, wherein the optical pulses are at asingle characteristic wavelength.

(55) The method of (53) or (54), further comprising determining at leastone property of the sample based on the detected fluorescent lifetimes.

(56) The method of (55), further comprising producing an electronicimage of a region of the sample, and indicating the at least onecharacteristic that is based on fluorescent lifetime in the image.

(57) The method of any one of (38)-(52), further comprising illuminatinga sample with optical pulses from the semiconductor diode, anddiscriminating arrival times of photons scattered back from the sampleinto at least two time bins with a single photodetector during a singlecharge accumulation interval for the single photodetector.

(58) The method of (57), further comprising producing an electronic,three-dimensional image of the sample based upon the discriminatedarrival times.

(59) A fluorescent lifetime analysis system comprising a semiconductordiode configured to emit light, a driving circuit configured to apply abipolar current pulse to the semiconductor diode to produce an opticalpulse, an optical system arranged to deliver the optical pulse to asample, and a photodetector configured to discriminate photon arrivaltimes into at least two time bins during a single charge-accumulationinterval of the photodetector.

(60) The system of (59), further comprising a pulse generator arrangedto provide an electrical pulse to the current driving circuit, whereinthe current driving circuit is configured to apply a bipolar pulse tothe semiconductor diode responsive to receiving the electrical pulse.

(61) The system of (60), wherein the electrical pulse is a unipolarpulse having a duration between 50 ps and 2 ns.

(62) The system of (60) or (61), wherein the current driving circuitcomprises a transistor having a gate terminal coupled to an output fromthe pulse generator and having current-carrying terminals connectedbetween a terminal of the semiconductor diode and a reference potential.

(63) The system of (62), further comprising a first resistor and firstcapacitor connected in parallel between an anode and a cathode of thesemiconductor diode, and a second resistor and second capacitorconnected in parallel between a gate terminal of the transistor and areference potential.

(64) The system of any one of (59)-(63), wherein the semiconductor diodecomprises a laser diode or light-emitting diode.

(65) The system of any one of (59)-(63), further comprising multiplewire bonds connected to a terminal of the semiconductor diode.

(66) The system of any one of (59)-(63), wherein the optical pulse has afull-width-half-maximum duration between 50 ps and 500 ps.

(67) The system of any one of (59)-(63), wherein the optical pulse has acharacteristic wavelength selected from the following group: 270 nm, 280nm, 325 nm, 340 nm, 370 nm, 380 nm, 400 nm, 405 nm, 410 nm, 450 nm, 465nm, 470 nm, 490 nm, 515 nm, 640 nm, 665 nm, 808 nm, and 980 nm.

(68) The system of any one of (59)-(63), further comprising an array ofphotodetectors in which the photodetector is located, the array ofphotodetectors configured to time-bin fluorescence from the sampleduring a single charge-accumulation interval for the optical pulse.

(69) The system of (68), further comprising imaging optics locatedbetween the sample and the photodetector array, wherein the imagingoptics are arranged to form an image at the photodetector array of aregion of the sample illuminated by the optical pulse.

(70) The system of (69), wherein the image formed at the photodetectorarray is an image of a microscopic region of the sample.

(71) A pulsed optical source comprising a semiconductor diode configuredto emit light, a first logic gate configured to form a first pulse at anoutput of the first logic gate, a driving circuit coupled to the firstlogic gate, wherein the driving circuit is configured to receive thefirst pulse and apply a bipolar electrical pulse to the semiconductordiode to produce an optical pulse responsive to receiving the firstpulse.

(72) The pulsed optical source of (71), wherein the first pulse is aunipolar pulse.

(73) The pulsed optical source of (72), further comprising a fan-outgate and a delay element coupled to the first logic gate, wherein thedelay element delays at least one output from the fan-out gate.

(74) The pulsed optical source of (73), wherein the delay element isconfigured to vary a pulse length of the unipolar pulse in incrementsbetween 1 ps and 5 ps.

(75) The pulsed optical source of any one of (71)-(74), wherein thefirst logic gate is configured to form the first pulse from twodifferential clock signals.

(76) The pulsed optical source of any one of (71)-(75), wherein thebipolar electrical pulse comprises a first pulse having a firstmagnitude and first polarity that is followed by a second pulse ofopposite polarity having a second magnitude different from the firstmagnitude.

(77) The pulsed optical source of (76), wherein the second magnitude isbetween 25% and 90% of the first magnitude.

(78) The pulsed optical source of any one of (71)-(77), furthercomprising multiple wire bonds connected to a terminal of thesemiconductor diode.

(79) The pulsed optical source of any one of (75)-(78), furthercomprising a second logic gate configured to form a second pulse fromthe two differential clock signals, wherein the second logic gate isconnected in parallel with the first logic gate and an output of thesecond logic gate is coupled to the driving circuit.

(80) The pulsed optical source of any one of (71)-(79), furthercomprising a transistor within the driving circuit having currentcarrying terminals connected between the semiconductor diode and areference potential.

(81) The pulsed optical source of (80), wherein the optical pulse has aduration between 50 ps and 2 ns.

(82) A pulsed optical source comprising a semiconductor diode configuredto emit light, and a driving circuit that includes a transistor coupledto a terminal of the semiconductor diode, wherein the driving circuit isconfigured to receive a unipolar pulse and apply a bipolar electricalpulse to the semiconductor diode responsive to receiving the unipolarpulse, wherein the transistor is connected in parallel with thesemiconductor diode between a current source and a reference potential.

(83) The pulsed optical source of (82) optionally having features of anyone of (2)-(4), (15), and (22)-(30), excluding features of (1), furthercomprising a resistor and a capacitor connected in parallel between thesemiconductor diode and the reference potential.

(84) The pulsed optical source of (82) or (83), wherein the transistoris configured to be normally conducting and is pulsed off with theunipolar pulse.

(85) The pulsed optical source of any one of (82)-(84), furthercomprising a photodetector array having a plurality of pixels that areeach configured to discriminate photon arrival times into at least twotime bins during a single charge-accumulation interval, and an opticalsystem arranged to form an image of an object, that is illuminated bythe pulsed optical source, on the photodetector array.

(86) A pulsed optical source comprising a semiconductor diode configuredto emit light, and plural first circuit branches connected to a firstterminal of the semiconductor diode, each circuit branch comprising atransistor having its current-carrying terminals connected between areference potential and the first terminal of the semiconductor diode.

(87) The pulsed optical source of (86) optionally having features of anyone of (4), (15), (16) and (22)-(30), excluding features of (1), whereina first reference potential in a first circuit branch of the pluralfirst circuit branches has a different value from a second referencepotential in a second circuit branch of the plural first circuitbranches.

(88) The pulsed optical source of (86) or (87), wherein a firstreference potential in a first circuit branch of the plural firstcircuit branches has a positive value and a second reference potentialin a second circuit branch of the plural first circuit branches has anegative value.

(89) The pulsed optical source of any one of (86)-(88), furthercomprising in each circuit branch a resistor connected between acurrent-carrying terminal of the transistor and the reference potential.

(90) The pulsed optical source of any one of (86)-(89), furthercomprising in each circuit branch a capacitor connected between acurrent-carrying terminal of the transistor and a ground potential.

(91) The pulsed optical source of any one of (86)-(90), furthercomprising a photodetector array having a plurality of pixels that areeach configured to discriminate photon arrival times into at least twotime bins during a single charge-accumulation interval, and an opticalsystem arranged to form an image of an object, that is illuminated bythe pulsed optical source, on the photodetector array.

(92) A pulsed optical source comprising a radio-frequency amplifierproviding a signal and an inverted signal, a logic gate configured toreceive the signal and a phase-shifted inverted signal and output apulse and an inverted pulse, a combiner configured to combine the pulseand inverted pulse onto a common output, and a semiconductor diodecoupled to the common output and configured to produce an optical pulseresponsive to receiving the pulse and inverted pulse.

(93) The pulsed optical source of (92) optionally having features of anyone of (4), (15), (16) and (22)-(30), excluding features of (1), furthercomprising a variable attenuator arranged to attenuate the pulse or theinverted pulse.

(94) The pulsed optical source of (92) or (93), further comprising adelay element arranged to temporally delay the pulse or the invertedpulse.

(95) The pulsed optical source of any one of (92)-(94), furthercomprising a DC block connect to an input of the radio-frequencyamplifier.

(96) The pulsed optical source of any one of (92)-(95), furthercomprising a photodetector array having a plurality of pixels that areeach configured to discriminate photon arrival times into at least twotime bins during a single charge-accumulation interval, and an opticalsystem arranged to form an image of an object, that is illuminated bythe pulsed optical source, on the photodetector array.

(97) A pulsed optical source comprising a radio-frequency logic gateconfigured to receive a first signal and an inverted version of thefirst signal and output a pulse and an inverted version of the pulse,and a semiconductor diode connect to the radio-frequency logic gate andarranged to receive the pulse at a first terminal of the semiconductordiode and the inverted version of the pulse at a second terminal of thesemiconductor diode and emit an optical pulse.

(98) The pulsed optical source of (97) optionally having features of anyone of (4), (15), (16) and (22)-(30), excluding features of (1), furthercomprising a first amplifier arranged to receive a periodic signal andoutput the first signal and the inverted version of the first signal,and a phase shifter arranged to vary a phase of the first signal or theinverted version of the first signal.

(99) The pulsed optical source of (97) or (98), further comprising aphotodetector array having a plurality of pixels that are eachconfigured to discriminate photon arrival times into at least two timebins during a single charge-accumulation interval, and an optical systemarranged to form an image of an object, that is illuminated by thepulsed optical source, on the photodetector array.

VI. Conclusion

Having thus described several aspects of several embodiments of a pulsedlaser, it is to be appreciated that various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. While the present teachings have been described inconjunction with various embodiments and examples, it is not intendedthat the present teachings be limited to such embodiments or examples.On the contrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

While various inventive embodiments have been described and illustrated,those of ordinary skill in the art will readily envision a variety ofother means and/or structures for performing the function and/orobtaining the results and/or one or more of the advantages described,and each of such variations and/or modifications is deemed to be withinthe scope of the inventive embodiments described. More generally, thoseskilled in the art will readily appreciate that all parameters,dimensions, materials, and configurations described are meant to beexamples and that the actual parameters, dimensions, materials, and/orconfigurations will depend upon the specific application or applicationsfor which the inventive teachings is/are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific inventive embodimentsdescribed. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, inventiveembodiments may be practiced otherwise than as specifically describedand claimed. Inventive embodiments of the present disclosure may bedirected to each individual feature, system, system upgrade, and/ormethod described. In addition, any combination of two or more suchfeatures, systems, and/or methods, if such features, systems, systemupgrade, and/or methods are not mutually inconsistent, is includedwithin the inventive scope of the present disclosure.

Further, though some advantages of the present invention may beindicated, it should be appreciated that not every embodiment of theinvention will include every described advantage. Some embodiments maynot implement any features described as advantageous. Accordingly, theforegoing description and drawings are by way of example only.

Numerical values and ranges may be described in the specification andclaims as approximate or exact values or ranges. For example, in somecases the terms “about,” “approximately,” and “substantially” may beused in reference to a value. Such references are intended to encompassthe referenced value as well as plus and minus reasonable variations ofthe value. For example, a phrase “between about 10 and about 20” isintended to mean “between exactly 10 and exactly 20” in someembodiments, as well as “between 10±δ1 and 20±δ2” in some embodiments.The amount of variation δ1, δ2 for a value may be less than 5% of thevalue in some embodiments, less than 10% of the value in someembodiments, and yet less than 20% of the value in some embodiments. Inembodiments where a large range of values is given, e.g., a rangeincluding two or more orders of magnitude, the amount of variation δ1,δ2 for a value could be as high as 50%. For example, if an operablerange extends from 2 to 200, “approximately 80” may encompass valuesbetween 40 and 120 and the range may be as large as between 1 and 300.When exact values are intended, the term “exactly” is used, e.g.,“between exactly 2 and exactly 200.”

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The section headings used are for organizational purposes only and arenot to be construed as limiting the subject matter described in any way.

Also, the technology described may be embodied as a method, of which atleast one example has been provided. The acts performed as part of themethod may be ordered in any suitable way. Accordingly, embodiments maybe constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

All definitions, as defined and used, should be understood to controlover dictionary definitions, definitions in documents incorporated byreference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used in the specification andin the claims, unless clearly indicated to the contrary, should beunderstood to mean “at least one.”

The phrase “and/or,” as used in the specification and in the claims,should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used shall only be interpreted as indicating exclusive alternatives(i.e. “one or the other but not both”) when preceded by terms ofexclusivity, such as “either,” “one of,” “only one of,” or “exactly oneof.” “Consisting essentially of,” when used in the claims, shall haveits ordinary meaning as used in the field of patent law.

As used in the specification and in the claims, the phrase “at leastone,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made by one of ordinary skillin the art without departing from the spirit and scope of the appendedclaims. All embodiments that come within the spirit and scope of thefollowing claims and equivalents thereto are claimed.

What is claimed is:
 1. A pulsed optical source comprising: asemiconductor diode configured to emit light; and a driving circuit thatincludes a transistor coupled to a terminal of the semiconductor diode,wherein the driving circuit is configured to receive a unipolar pulseand apply a bipolar electrical pulse to the semiconductor dioderesponsive to receiving the unipolar pulse.
 2. The pulsed optical sourceof claim 1, wherein the bipolar electrical pulse comprises a first pulsehaving a first magnitude and first polarity that is followed by a secondpulse of opposite polarity having a second magnitude different from thefirst magnitude.
 3. The pulsed optical source of claim 2, wherein thesecond magnitude is between 25% and 90% of the first magnitude.
 4. Thepulsed optical source of claim 1, further comprising multiple wire bondsconnected to a terminal of the semiconductor diode.
 5. The pulsedoptical source of claim 1, further comprising a pulse generator coupledto the driving circuit and configured to form the unipolar pulse andoutput the unipolar pulse to the driving circuit.
 6. The pulsed opticalsource of claim 5, wherein a pulse length of the unipolar pulse isbetween 50 ps and 500 ps.
 7. The pulsed optical source of claim 5,wherein the pulse generator comprises a first logic gate that forms theunipolar pulse from two differential clock signals.
 8. The pulsedoptical source of claim 7, wherein the pulse generator further comprisesan adjustable delay element configured to vary a pulse length of theunipolar pulse in increments between 1 ps and 5 ps.
 9. The pulsedoptical source of claim 7, wherein the transistor has current-carryingterminals connected between a cathode of the semiconductor diode and areference potential and has a gate terminal coupled to the first logicgate.
 10. The pulsed optical source of claim 9, further comprising acapacitor connected between the gate terminal of the transistor and anoutput from the first logic gate.
 11. The pulsed optical source of claim9, wherein the transistor comprises a high-electron-mobilityfield-effect transistor.
 12. The pulsed optical source of claim 9,wherein the transistor is configured to switch up to 4 amps through thesemiconductor diode for a duration between 50 ps and 2 ns.
 13. Thepulsed optical source of claim 9, further comprising a second logic gateconnected in parallel with the first logic gate and arranged to form asecond unipolar pulse from the two differential clock signals, whereinan output from the second logic gate is coupled to the gate terminal ofthe transistor.
 14. The pulsed optical source of claim 9, wherein adrain terminal of the transistor connects directly to a cathode of thesemiconductor diode.
 15. The pulsed optical source of claim 14, furthercomprising a first capacitor and resistor connected in parallel to thedrain terminal.
 16. The pulsed optical source of claim 14, furthercomprising a second capacitor connected between an anode of thesemiconductor diode and a source terminal of the transistor.
 17. Thepulsed optical source of claim 1, wherein an optical pulse having afull-width-half maximum duration between 50 ps and 500 ps is emittedfrom the semiconductor diode responsive to application of the bipolarelectrical pulse.
 18. The pulsed optical source of claim 1, wherein theoptical pulse has a characteristic wavelength selected from thefollowing group: 270 nm, 280 nm, 325 nm, 340 nm, 370 nm, 380 nm, 400 nm,405 nm, 410 nm, 450 nm, 465 nm, 470 nm, 490 nm, 515 nm, 640 nm, 665 nm,808 nm, and 980 nm.
 19. The pulsed optical source of claim 17, wherein atail of the optical pulse remains below at least 20 dB from the peak ofthe pulse after 250 ps from the peak of the pulse.
 20. The pulsedoptical source of claim 1, further comprising a saturable absorberarranged to receive an optical pulse from the semiconductor diode. 21.The pulsed optical source of claim 1, further comprising: aphotodetector array having a plurality of pixels that are eachconfigured to discriminate photon arrival times into at least two timebins during a single charge-accumulation interval; and an optical systemarranged to form an image of an object, that is illuminated by thepulsed optical source, on the photodetector array.
 22. A fluorescentlifetime analysis system comprising: a driving circuit configured toapply a bipolar current pulse to a semiconductor diode to produce anoptical pulse that is delivered to a sample; and a photodetectorconfigured to discriminate photon arrival times into at least two timebins during a single charge-accumulation interval of the photodetector.23. The system of claim 22, further comprising a pulse generatorarranged to provide an electrical pulse to the current driving circuit,wherein the current driving circuit is configured to apply a bipolarpulse to the semiconductor diode responsive to receiving the electricalpulse.
 24. The system of claim 23, wherein the electrical pulse is aunipolar pulse having a duration between 50 ps and 2 ns.
 25. The systemof claim 23, wherein the current driving circuit comprises a transistorhaving a gate terminal coupled to an output from the pulse generator andhaving current-carrying terminals connected between a terminal of thesemiconductor diode and a reference potential.
 26. The system of claim25, further comprising: a first resistor and first capacitor connectedin parallel between an anode and a cathode of the semiconductor diode;and a second resistor and second capacitor connected in parallel betweena gate terminal of the transistor and a reference potential.
 27. Thesystem of claim 22, further comprising multiple wire bonds connected toa terminal of the semiconductor diode.
 28. The system of claim 22,wherein the optical pulse has a full-width-half-maximum duration between50 ps and 500 ps.
 29. The system of claim 22, wherein the optical pulsehas a characteristic wavelength selected from the following group: 270nm, 280 nm, 325 nm, 340 nm, 370 nm, 380 nm, 400 nm, 405 nm, 410 nm, 450nm, 465 nm, 470 nm, 490 nm, 515 nm, 640 nm, 665 nm, 808 nm, and 980 nm.30. The system of claim 22, further comprising an array ofphotodetectors in which the photodetector is located, the array ofphotodetectors configured to time-bin fluorescence from the sampleduring a single charge-accumulation interval for the optical pulse. 31.The system of claim 30, further comprising imaging optics locatedbetween the sample and the photodetector array, wherein the imagingoptics are arranged to form an image at the photodetector array of aregion of the sample illuminated by the optical pulse.
 32. The system ofclaim 31, wherein the image formed at the photodetector array is animage of a microscopic region of the sample.